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1
Sipert, Carla Renata. "Produção de MIP-1alfa e SDF-1 por fibroblastos de polpa dental humana em cultura frente ao desafio com Enterococcus faecalis inativado por calor." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/25/25138/tde-15102008-164844/.
Full textAbstract:
A polpa dental é formada de tecido conjuntivo frouxo sendo constituída por diversas células, dentre as quais os fibroblastos são as mais numerosas. Ao serem submetidas a agressões diversas, estas células respondem com a liberação de substâncias, tais como citocinas e quimiocinas, que participam de maneira ativa no processo inflamatório. Assim sendo, este trabalho teve como proposição: 1. avaliar a capacidade de fibroblastos de polpa dental humana em cultura em produzirem as quimiocinas MIP-l\'alfa\' /CCL3 e SDF-1/CXCL12; 2. avaliar a produção destas quimiocinas pelos fibroblastos quando estimulados por Enterococcus faecalis morto por calor com relação à quantidade de bactérias por célula e 3. avaliar a liberação destas quimiocinas com relação ao tempo de estímulo. Para o estabelecimento das culturas, foi coletada a polpa de terceiro molar hígido de um paciente saudável. O tecido foi extraído, armazenado e picotado em meio de cultura para fibroblastos (DMEM), os quais foram utilizados a partir da quarta passagem. Após adesão das células a placas de 24 poços, o meio de cultura contendo Enterococcus .faecalis morto por calor numa concentração correspondente a 1, 10 e 100 bactérias por fibroblasto foi adicionado aos poços. Após 1, 6 e 24 horas, o sobrenadante das células foi coletado para a análise por ELISA. A análise estatística foi realizada aplicando-se o teste Kruskal-Wallis com nível de significância de 5%. A produção de MIP-l\'alfa\' /CCL3 e SDF-l/CXCL12 pelas células pôde ser detectada por ELISA. Os fibroblastos pulpares se mostraram capazes de produzir SDF-1 constitutivamente sendo que o estímulo bacteriano levou a uma diminuição estatisticamente significativa desta produção. A produção de MIP-l\'alfa\' também foi detectada tanto de maneira constitutiva como em resposta ao desafio microbiano. Enquanto a concentração intermediária de bactéria por fibroblasto (10:1) mostrou uma produção semelhante ao grupo controle, as concentrações de 1 e 100 bactérias por fibroblasto induziram aumento maior na primeira hora de estímulo. Essas diferenças, entretanto, não foram estatisticamente significativas. A capacidade dos fibroblastos secretarem quimiocinas, como MIP-l\'alfa\' e SDF-1, reforça a importância dessas células dentro do contexto de imunidade e inflamação pulpar, principalmente por serem as células mais numerosas deste microambiente.Dental pulp is a connective tissue structure constituted by many different cell types. Among them, the fibroblasts are the most frequent ones. When challenged by different aggressive agents, these cells are able to release some substances like cytokines and chemokines, which are essential to trigger the inflammatory process. The aims of this study were: 1. to evaluate the ability of fibroblasts to produce the chemokines MIP-l\'alfa\'/CCL3) and SDF-1/CXCL12; 2. to evaluate the expression of these chemokines by fibroblasts when challenged by heat killed Enterococcus. faecalis in gradual concentrations and 3. to evaluate the production of these chemokines in a time course manner. The dental pulp from non-carious third molar was collected from a healthy patient. Explants were made and stocked in culture medium (DMEM) for fibroblasts growth. The cells were used since passage four. In a 24-well plate and after reaching confluence, culture medium alone or containing heat killed E. faecalis at proportion 1:1, 10:1 and 100:1 bacteria:fibroblast, were added to the fibroblasts. After 1, 6 and 24 hours, the supernatants were collected for analysis. The protein detection of MIP-l\'alfa\'/CCL3 and SDF-1/CXCL12 was performed by ELISA. For statistical analysis, data were assessed by Kruskal-Wallis followed by Miller post-test. Significance levels of 5% were adopted. Production of both chemokines was detected by ELISA. Pulp fibroblasts were able to produce SDF-1 constitutively. This production decreased with the increase in the number of heat killed E. faecalis increased (p < 0.05). Production of MIP-l\'alfa\' was detected in unchallenged and challenged cells. The median bacterial concentration (10:1) presented a profile production similar to that of unstimulated cells. Bacterial concentrations of 1 and 100 microrganisms/cell showed a highly enhanced production of MIP-l\'alfa\' at the first hour of stimulum; however, these data were not statistically significant (p > 0.05). Fibroblasts ability to produce chemokines, like MIP-l\'alfa\' and SDF-1, confirms their importance at immune and inflammatory events in dental pulp, specially being fibroblasts the most abundant cells at this microenvironment .
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Kashpur, Olga. "Oxygen-mediated basic fibroblast growth factor (FGF2) effects on adult human dermal fibroblasts." Digital WPI, 2015. https://digitalcommons.wpi.edu/etd-dissertations/546.
Full textAbstract:
This thesis investigates the effects of low oxygen culture conditions and fibroblast growth factor-2 (FGF2) on adult human dermal fibroblasts.
It was previously shown that low oxygen and FGF2 culture conditions lead to an extension of proliferative lifespan, low-level activation of stem cell genes, and global transcriptional changes in adult human dermal fibroblasts. Additionally, an increased in vivo tissue regenerative response can be observed when human muscle-derived fibroblasts grown with FGF2 and low oxygen are implanted into mouse muscle injury, leading to a decrease in collagen deposition and scar formation and increase of functional skeletal muscle regeneration, including formation of Pax7+ muscle stem cells.
These findings led to an analysis of key cellular oxygen sensors, hypoxia inducible factors (HIFs) and their role in this regenerative response. Directly linking these factors with the regenerative response, I have shown, with knockdown experiments, that HIF-2α is required for the increased proliferative capability and decreased senescence of human dermal fibroblasts (hDFs) induced by hypoxia. I have also determined that low oxygen causes an early and transient increase of HIF-1α and late and sustained increase of HIF-2α protein accompanied by increased nuclear translocation. Using overexpression and knockdown approaches via lent-virus, I determined that HIF-2α appears to modulate FGF2 signaling through the FGF receptors. First, under low oxygen conditions, exogenous FGF2 led to downregulation of endogenous FGF2, which can be mimicked by overexpression of HIF-2α. In ambient oxygen we didn't see this effect. Second, HIF-2α overexpression appears to lead to increases in FGFR1 phosphorylation and consequently increased ERK1/2 phosphorylation, and increases in the expression of heparan sulfate modifying enzymes (NDST1, NDST2, and EXTL2). Lastly, sustained supplementation with FGF2 in low oxygen inhibits receptor-mediated FGF2 signaling.
To understand these effects at the transcriptional level, using microarray technology, we identified oxygen-mediated FGF2 effects on genes involved in cell survival and proliferation.
Through bioinformatics analyses, I determined that genes involved in wound healing (extracellular matrix genes, adhesion molecules, cytokines) are upregulated in FGF2 treated fibroblasts grown under low oxygen. By utilizing a gain-of-function approach, we were able to assess the effects of altered HIF-2α activity on the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 in adult hDFs. The results indicate that overexpression of the HIF-2α transcription factor increases Oct4 mRNA, but not Oct4 protein, levels, and had no effect on Nanog and Lin28 proteins. HIF-2α overexpression also mediated FGF2 induction of Sox2 and Rex1 proteins of higher molecular weight.
This thesis expands our knowledge about effects of low oxygen and FGF2 on adult human dermal fibroblasts and explains in part, how FGF2 under low oxygen conditions may lead to increased proliferation, extended life span, regenerative competency and increased developmental plasticity of adult hDFs.
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Grandi, Fabrizio [UNESP]. "Fibroblastos associados ao câncer e correlação com parãmetros patológicos em melanomas cutâneos caninos." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/95886.
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grandi_f_me_botfm.pdf: 914284 bytes, checksum: 7e8beca0af748a53dac236ca291fea62 (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O melanoma é uma das neoplasias cutâneas mais comumente diagnosticadas no homem e nos cães. O microambiente tumoral é composto pelas células neoplásicas e células estromais interagindo constantemente para garantir a progressão tumoral. Os fibroblastos associados ao câncer (FAC) representam uma população heterogênea de células caracterizadas pela expressão de diversos marcadores incluindo a proteína α-SMA e S100A4. Acredita-se que estas células originem-se de diversas fontes incluindo a transição endotelial fibroblástica. Neste trabalho, quantificamos a imunoexpressão da prpteína S100A4 nos fibroblastos associados ao câncer em melanomas cutâneos caninos, verificamos a potencial contribuição das células endoteliais na gênese desta população e correlacionamos os achados com parâmetros patológicos, incluindo a microdensidade vascular. Quarenta e oito casos de melanoma dermais caninos (21 epitelióides, 14 fusiformes e 13 mistos) previamente categorizados nos níveis de Clark 4, 5 e até 4 foram submetidos a imunofluorescência dupla utilizando os anticorpos primários α-SMA, fator de Von Willebrand (vWF) e S100A4 objetivando-se caracterizar os fibroblastos associados ao câncer e a contribuição da transição endotelial mesenquimal. Os melanomas não pigmentados foram caracterizados pela imunoistoquímica utilizando-se os anticorpos vimentina, pancitoqueratina, S100 e Melan A. A densidade microvascular foi analisada utilizando-se a técnica de imunofluorescência e o anticorpo primário anti-fator de Von Willebrand. O cálculo do número de vasos foi realizado selecionando-se cinco campos microscópicos de 200x contendo o maior número de vasos. O percentul de expressão da proteína S100A4 foi calculado através da técnica de segmentação de conglomerados de cor. Apenas um caso demonstrou...
Skin melanoma is one of the most common skin neoplasm seen in humans and dogs. Tumor microenvironment is composed by cancer cells and stromal cells that interacts to guarantee tumor progression. Cancer associated fibroblasts (CAF) represents a heterogeneous cell population characterized by expression of several markers including α-SMA and S100A4 proteins. These cells are thought to derive from different sources including endothelial-to-fibroblast transition. Here we characterize CAF in canine skin melanomas, verify the potential contribution of the endothelial cells to this population and correlate findings to pathological parameters, including microvascular density (MVD). Forth-eight cases of canine dermal melanomas (21 epithelioid, 14 spindle and 13 mixed) classified under Clark´s level 4 and 5 were submitted to a double immunofluorescence assay using primary antibodies α-SMA, Von Willebrand Factor (vWF) and S100A4 in order to characterize cancer associated fibroblasts and verify the contribution of endothelial to fibroblast transition. Non-pigmented samples were characterized by immunohistochemistry using primary antibodies pan-cytokeratin, vimentin, S100 and Melan A. Microvascular density was evaluated by immunofluorescent assay using vWF and by calculating total number of vessels in five 200x fields (“hotspots”).S100A4 imunoexpression was calculated using K-means clustering segmentation method. Only one case showed α-SMA and vWF co-expression restricted to myofibroblasts in tumor stroma. The cells were predominantly peritumoral and periadnexal. S100A4 expression was significantly different among three histotypes with mixed melanomas displaying lesser percentage of positive cells. Some neoplastic cells mainly in spindle cell melanomas were also positive for S100A4. There were no significant differences between MVD/histotypes... (Complete abstract click electronic access below)
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Silva, Rubia Bueno da [UNESP]. "Efeitos dos fatores de crescimento fibroblástico 10 e 18 (FGFs 10 e 18) sobre a esteroidogênese em ovários fetais bovinos." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/105894.
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silva_rb_dr_botfmvz.pdf: 496820 bytes, checksum: 055e35414ee23830cc537670a4ae215b (MD5)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Durante o desenvolvimento ovariano fetal, a formação folicular inicial é decisiva para a fertilidade da fêmea, pois define sua reserva gametogênica. Tem sido proposto que a progesterona e o estradiol desempenham papel regulatório na foliculogênese pré-antral, de forma que sua produção reduzida em ovários fetais bovinos antecede o surgimento de folículos primordiais e primários. Recentemente, os FGFs 10 e 18 foram reportados em folículos ovarianos bovinos como redutores dos níveis de esteróides, o que parece envolver a inibição da expressão de enzimas necessárias à esteroidogênese. Em adição, a expressão do FGF10 foi observada durante o desenvolvimento ovariano fetal bovino, e esteve positivamente associada ao aumento no número de folículos primários. O presente estudo investigou primeiramente o padrão de expressão do RNAm das enzimas esteroidogênicas (StAR, CYP11A1, 3β-HSD, CYP17A1, CYP19A1 e 17β-HSD) em ovários de fetos bovinos em idades gestacionais específicas (60, 75, 90, 120, 150 e 210 dias). Todos os genes investigados se mostraram expressos e regulados ao longo da gestação. Os níveis de RNAm da CYP19A1 diminuíram dos 60 para os 90 dias, sugerindo envolvimento desta enzima com a produção decrescente de estradiol observada previamente durante este período gestacional. A expressão das demais enzimas foi elevada ao longo da gestação, coincidente com o aumento da competência esteroidogênica descrito preliminarmente durante o desenvolvimento folicular inicial. Em adição, foi investigada a participação dos FGFs 10 e 18 na esteroidogênese ovariana fetal bovina. A expressão do FGF18 e de seus receptores (FGFR2C, FGFR3C e FGFR4) foi detectada em ovários fetais bovinos ao longo da gestação (60, 75, 90, 120, 150 e 210 dias). A abundância de RNAm do FGF18 aumentou...
During fetal ovarian development, early follicular formation is essential to female fertility, when the gametogenic reserve is defined. It has been proposed that progesterone and estradiol play regulatory role on preantral folliculogenesis, once its reduced production in bovine fetal ovaries precedes primordial and primary follicle assembly. Recentlly, FGFs 10 and 18 were reported in bovine ovarian follicles as reducers of steroids levels, and this seems to involve the inhibition of enzymes necessary to steroidogenesis. In addition, FGF10 expression was observed during bovine fetal ovary development, and it was positively associated with the elevation on primary follicles number. The present study first investigated the mRNA expression patterns for steroidogenic enzymes (StAR, CYP11A1, HSD3B1, CYP17A1, CYP19A1 and HSD17B1) in bovine fetal ovaries at specific gestational ages (60, 75, 90, 120, 150 e 210 days). Expression of all investigated genes was detected and regulated through gestation. Messenger RNA levels of CYP19A1 decreased from days 60 to 90 of gestation, suggesting involvement of this enzyme on decrescent estradiol production previously observed during this gestational period. The expression of other enzymes was elevated during gestational period, which was coincident with the enhance of steroidogenic competence previously described during early follicular development. In addition, the participation of FGFs 10 and 18 on steroidogenesis in bovine fetal ovaries was investigated. The expression of FGF18 and its receptors (FGFR2C, FGFR3C and FGFR4) was detected in bovine fetal ovaries through gestation (60, 75, 90, 120, 150 e 210 days). The mRNA abundance of FGF18 enhanced between 90 and 120 days and decreased at 210 days. The expression of FGFR2C and FGFR4 did not vary during... (Complete abstract click electronic access below)
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Silva, Rubia Bueno da. "Efeitos dos fatores de crescimento fibroblástico 10 e 18 (FGFs 10 e 18) sobre a esteroidogênese em ovários fetais bovinos /." Botucatu, 2012. http://hdl.handle.net/11449/105894.
Full textAbstract:
Orientador: José Buratini JuniorBanca: José Antonio Visitin
Banca: Guilherme de Paula Nogueira
Banca: Fernanda da Cruz Landim e Alvarenga
Banca: Sony Dimas Bicudo
Resumo: Durante o desenvolvimento ovariano fetal, a formação folicular inicial é decisiva para a fertilidade da fêmea, pois define sua reserva gametogênica. Tem sido proposto que a progesterona e o estradiol desempenham papel regulatório na foliculogênese pré-antral, de forma que sua produção reduzida em ovários fetais bovinos antecede o surgimento de folículos primordiais e primários. Recentemente, os FGFs 10 e 18 foram reportados em folículos ovarianos bovinos como redutores dos níveis de esteróides, o que parece envolver a inibição da expressão de enzimas necessárias à esteroidogênese. Em adição, a expressão do FGF10 foi observada durante o desenvolvimento ovariano fetal bovino, e esteve positivamente associada ao aumento no número de folículos primários. O presente estudo investigou primeiramente o padrão de expressão do RNAm das enzimas esteroidogênicas (StAR, CYP11A1, 3β-HSD, CYP17A1, CYP19A1 e 17β-HSD) em ovários de fetos bovinos em idades gestacionais específicas (60, 75, 90, 120, 150 e 210 dias). Todos os genes investigados se mostraram expressos e regulados ao longo da gestação. Os níveis de RNAm da CYP19A1 diminuíram dos 60 para os 90 dias, sugerindo envolvimento desta enzima com a produção decrescente de estradiol observada previamente durante este período gestacional. A expressão das demais enzimas foi elevada ao longo da gestação, coincidente com o aumento da competência esteroidogênica descrito preliminarmente durante o desenvolvimento folicular inicial. Em adição, foi investigada a participação dos FGFs 10 e 18 na esteroidogênese ovariana fetal bovina. A expressão do FGF18 e de seus receptores (FGFR2C, FGFR3C e FGFR4) foi detectada em ovários fetais bovinos ao longo da gestação (60, 75, 90, 120, 150 e 210 dias). A abundância de RNAm do FGF18 aumentou... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: During fetal ovarian development, early follicular formation is essential to female fertility, when the gametogenic reserve is defined. It has been proposed that progesterone and estradiol play regulatory role on preantral folliculogenesis, once its reduced production in bovine fetal ovaries precedes primordial and primary follicle assembly. Recentlly, FGFs 10 and 18 were reported in bovine ovarian follicles as reducers of steroids levels, and this seems to involve the inhibition of enzymes necessary to steroidogenesis. In addition, FGF10 expression was observed during bovine fetal ovary development, and it was positively associated with the elevation on primary follicles number. The present study first investigated the mRNA expression patterns for steroidogenic enzymes (StAR, CYP11A1, HSD3B1, CYP17A1, CYP19A1 and HSD17B1) in bovine fetal ovaries at specific gestational ages (60, 75, 90, 120, 150 e 210 days). Expression of all investigated genes was detected and regulated through gestation. Messenger RNA levels of CYP19A1 decreased from days 60 to 90 of gestation, suggesting involvement of this enzyme on decrescent estradiol production previously observed during this gestational period. The expression of other enzymes was elevated during gestational period, which was coincident with the enhance of steroidogenic competence previously described during early follicular development. In addition, the participation of FGFs 10 and 18 on steroidogenesis in bovine fetal ovaries was investigated. The expression of FGF18 and its receptors (FGFR2C, FGFR3C and FGFR4) was detected in bovine fetal ovaries through gestation (60, 75, 90, 120, 150 e 210 days). The mRNA abundance of FGF18 enhanced between 90 and 120 days and decreased at 210 days. The expression of FGFR2C and FGFR4 did not vary during... (Complete abstract click electronic access below)
Doutor
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Grandi, Fabrizio. "Fibroblastos associados ao câncer e correlação com parãmetros patológicos em melanomas cutâneos caninos /." Botucatu, 2012. http://hdl.handle.net/11449/95886.
Full textAbstract:
Orientador: Noeme Sousa RochaBanca: Hélio Amante Miot
Banca: Bruno Cogliati
Resumo: O melanoma é uma das neoplasias cutâneas mais comumente diagnosticadas no homem e nos cães. O microambiente tumoral é composto pelas células neoplásicas e células estromais interagindo constantemente para garantir a progressão tumoral. Os fibroblastos associados ao câncer (FAC) representam uma população heterogênea de células caracterizadas pela expressão de diversos marcadores incluindo a proteína α-SMA e S100A4. Acredita-se que estas células originem-se de diversas fontes incluindo a transição endotelial fibroblástica. Neste trabalho, quantificamos a imunoexpressão da prpteína S100A4 nos fibroblastos associados ao câncer em melanomas cutâneos caninos, verificamos a potencial contribuição das células endoteliais na gênese desta população e correlacionamos os achados com parâmetros patológicos, incluindo a microdensidade vascular. Quarenta e oito casos de melanoma dermais caninos (21 epitelióides, 14 fusiformes e 13 mistos) previamente categorizados nos níveis de Clark 4, 5 e até 4 foram submetidos a imunofluorescência dupla utilizando os anticorpos primários α-SMA, fator de Von Willebrand (vWF) e S100A4 objetivando-se caracterizar os fibroblastos associados ao câncer e a contribuição da transição endotelial mesenquimal. Os melanomas não pigmentados foram caracterizados pela imunoistoquímica utilizando-se os anticorpos vimentina, pancitoqueratina, S100 e Melan A. A densidade microvascular foi analisada utilizando-se a técnica de imunofluorescência e o anticorpo primário anti-fator de Von Willebrand. O cálculo do número de vasos foi realizado selecionando-se cinco campos microscópicos de 200x contendo o maior número de vasos. O percentul de expressão da proteína S100A4 foi calculado através da técnica de segmentação de conglomerados de cor. Apenas um caso demonstrou... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Skin melanoma is one of the most common skin neoplasm seen in humans and dogs. Tumor microenvironment is composed by cancer cells and stromal cells that interacts to guarantee tumor progression. Cancer associated fibroblasts (CAF) represents a heterogeneous cell population characterized by expression of several markers including α-SMA and S100A4 proteins. These cells are thought to derive from different sources including endothelial-to-fibroblast transition. Here we characterize CAF in canine skin melanomas, verify the potential contribution of the endothelial cells to this population and correlate findings to pathological parameters, including microvascular density (MVD). Forth-eight cases of canine dermal melanomas (21 epithelioid, 14 spindle and 13 mixed) classified under Clark's level 4 and 5 were submitted to a double immunofluorescence assay using primary antibodies α-SMA, Von Willebrand Factor (vWF) and S100A4 in order to characterize cancer associated fibroblasts and verify the contribution of endothelial to fibroblast transition. Non-pigmented samples were characterized by immunohistochemistry using primary antibodies pan-cytokeratin, vimentin, S100 and Melan A. Microvascular density was evaluated by immunofluorescent assay using vWF and by calculating total number of vessels in five 200x fields ("hotspots").S100A4 imunoexpression was calculated using K-means clustering segmentation method. Only one case showed α-SMA and vWF co-expression restricted to myofibroblasts in tumor stroma. The cells were predominantly peritumoral and periadnexal. S100A4 expression was significantly different among three histotypes with mixed melanomas displaying lesser percentage of positive cells. Some neoplastic cells mainly in spindle cell melanomas were also positive for S100A4. There were no significant differences between MVD/histotypes... (Complete abstract click electronic access below)
Mestre
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Bedran, Telma Blanca Lombardo [UNESP]. "Efeito antimicrobiano e modulador da resposta imune dos peptídeos hBD-3 e LL-37 e dos polifenóis o chá verde e do cranberry." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/124090.
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000830081.pdf: 809941 bytes, checksum: ea53cd5ef0cec7fb993a2745e82dad53 (MD5)The antimicrobial peptides LL-37, hBD-1, hBD-2 and hBD-3 are considered an endogenous antibiotic, with important role in the prevention of periodontal diseases due to their ability to regulate the immune response. However those peptides could be degraded by periodontal pathogens. Therefore, therapies able to up regulate the secretion of those peptides by human cells, and the association of antimicrobial peptides with natural compounds, which may act in synergism to modulate the immune response, may be a novel approach for effectively controlling periodontal diseases. The aim of this in vitro study were: i) investigate the ability of green tea extract and EGCG to induce hBD-1 and hBD-2 secretion and gene expression by gingival epithelial cells (B11) and to protect hBDs from degradation by P. gingivalis, ii) A 3D co-culture model of gingival epithelial cells and fibroblasts stimulated with A. actinomycetemcomitans LPS (1 μg/ml) were used to investigated the anti-inflammatory properties of the hBD-3, LL-37, ACPACs and EGCG and to determine whether LL-37 acts in synergy with AC-PACs, EGCG and hBD-3. Gingival epithelial cells were stimulated with green tea extract or EGCG in the presence and absence of specific inhibitors. The secretion and gene expression of hBD-1 and hBD-2 was respectively measured by ELISA and qPCR. The ability of green tea extract and EGCG to prevent hBDs degradation by P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA. A 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to A. actinomycetemcomitans LPS stimulation compared to fibroblasts and epithelial cells individually. The 3D co-culture model was stimulated with noncytotoxic concentrations of: i) hBD-3 (10 and 20 μM) ...(Complete abstract electronic access below)
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Santos, Carolina Carvalho de Oliveira 1984. "Expressão e produção de IL-6, TNF-'alfa' e MCP-1 por fibroblastos (3T3) e odontoblastos (MDPC-23) após exposição a bactérias orais." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/289724.
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Orientador: Alexandre Augusto ZaiaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: A polpa dentária contém uma heterogeneidade de células, dentre elas destacam-se os fibroblastos e odontoblastos. Quando este tecido é agredido por micro-organismos ou seus bioprodutos, fibroblastos e odontoblastos secretam diversas citocinas inflamatórias para ativação do sistema imune, dentre estas TNF-'alfa', IL-6 e MCP-1. Este estudo avaliou a expressão gênica e a produção de citocinas inflamatórias após o contato in vitro das espécies Sreptococcus mutans, Porphyromonas gingivalis, Enterococcus faecalis e seus bioprodutos com odontoblastos e fibroblastos. A expressão gênica foi avaliada por RT-qPCR e as proteínas foram quantificadas por meio de citometria de fluxo, tipo CBA, nos períodos de 4, 8 e 24 horas. As bactérias estudadas promoveram sensibilização das células para expressão e produção das citocinas IL-6, TNF-'alfa', e MCP-1 principalmente nas primeiras 8 horas de contato. Tanto o contato direto com as bactérias como o contato com seus bioprodutos resultaram em estímulo para as células, contudo, o contato direto com as bactérias S. mutans e E. faecalis se mostrou mais eficiente para a estimulação de expressão e produção de citocinas. Por outro lado, o contato indireto com P. gingivalis mostrou ser mais efetivo para estimular a produção das citocinas. Desta forma, pode-se concluir que odontoblastos e fibroblastos são capazes de expressar e produzir citocinas pró-inflamatórias a partir de diferentes contatos quando estimulados por tipos bacterianos distintos.
Abstract: Dental pulp contains a heterogeneity of cells in its composition, among them stand out from fibroblasts and odontoblasts. When this tissue is attacked by bacteria or their by-products, fibroblasts and odontoblasts can secrete several inflammatory cytokines in order to attract and activate immune system to contain infectious agents. TNF-'alfa', IL-6 and MCP-1 are proteins secreted by fibroblasts and odontoblasts mainly involved in the origin and activation of the inflammatory process. This study aimed to evaluate gene expression and secretion of inflammatory cytokines after in vitro contact of bacteria Sreptococcus mutans, Porphyromonas gingivalis, Enterococcus. faecalis and its by-products with odontoblasts and fibroblasts. The proteins were quantified by flow cytometry type CBA and gene expression by Real Time - PCR in periods of 4, 8 and 24 hours. The results showed that the studied bacteria promoted sensitizing cells for expression and production of the cytokines IL-6, TNF-'alfa', MCP-1, particularly during the first 8 hours of contact. Both direct contact with bacterias or their by-products resulting in stimulation to the cells, however, direct contact with the bacteria S. mutans and E. faecalis was more efficient for stimulating expression and cytokine production. On the other hand, the indirect contact with P. gingivalis shown to be more effective to stimulate the production of cytokines. Thus, we may conclude that fibroblasts and odontoblasts are able to express and produce proinflammatory cytokines from different contacts when stimulated by different bacterial types
Doutorado
Endodontia
Doutora em Clínica Odontológica
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Silva, Giuliana Thaíse Araújo da. "Influência na variação da potência de irradiação por LED com tempo fixo em cultura fibroblástica L929." Universidade Federal de Goiás, 2018. http://repositorio.bc.ufg.br/tede/handle/tede/8540.
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The Light Emitting Diode (LED) is a semiconductor device that emits light source and can be used as a tissue modeler by means of the transformation of received photons. Photobiomodulation therapy (PBMT) is a specific term for the therapeutic application of light and acts as therapy, analgesia and anti-inflammatory. The objective of this work was to verify the effectiveness of LED at fixed time in the stimulation of fibroblasts. For this purpose, the L929 cell line submitted to LED irradiation (630 nm) was used, in triplicate, at the potency of 50, 75 and 100 mW, for five seconds and compared with non-irradiated control group. Next, cell viability, proliferation, nitric oxide production and collagen synthesis were observed. The results revealed that there was no cytotoxicity after 24, 48 and 72 hours of irradiation; there was an increase in the production of nitrite between the group of 72 hours and the other experimental groups and increase in the synthesis of collagen, directly proportional to the potencies used. Thus the irradiation allowed the fibroblastic stimulation with increased activity in the healing process.
O Diodo Emissor de Luz (Light Emissor Diode - LED) é um dispositivo semicondutor que emite fonte de luz e pode ser usado como modelador tecidual por meio da transformação de fótons recebidos. A fotobiomodulação (Photobiomodulation therapy - PBMT) é um termo específico para aplicação terapêutica da luz e atua como analgesia e anti-inflamatório. O objetivo deste trabalho foi de verificar a efetividade do LED em tempo fixo na estimulação de fibroblastos. Para tanto, utilizou-se a linhagem celular L929 submetida à irradiação LED (630 nm), em triplicata, nas potências de 50, 75 e 100 mW, por cinco segundos e comparou-se com grupo controle não irradiado. Em seguida, observou-se a viabilidade celular, proliferação, produção de óxido nítrico e síntese de colágeno. Os resultados revelaram que não houve citotoxicidade após 24, 48 e 72 horas de irradiação; houve aumento na produção de nitrito entre o grupo de 72 horas e os outros grupos experimentais e aumento na síntese de colágeno, diretamente proporcionais às potências utilizadas. Assim a irradiação possibilitou a estimulação fibroblástica com aumento de sua atividade no processo de cicatrização.
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Nogueira, Alessandra Fonseca Gambini. "Estudo in vitro do efeito de cones de obturação endodôntica na biomodulação de fibroblastos de ligamento periodontal." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/23/23156/tde-27112017-122839/.
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A obturação do canal radicular é uma etapa fundamental para o sucesso do tratamento endodôntico. É desejável que os materiais empregados nesta fase não interferiram negativamente com o reparo tecidual, mas preferencialmente estimulem a regeneração dos tecidos periapicais. Recentemente, cones de guta-percha combinados com material biocerâmico foram desenvolvidos com esta finalidade. Sendo assim, o objetivo deste estudo foi investigar o potencial citotóxico e biomodulador de cones de guta-percha convencionais, de cones de guta-percha contendo biocerâmica e de cones de polímero sobre células de ligamento periodontal in vitro. Cultura de fibroblastos de ligamento periodontal foi estabelecida a partir de terceiro molar humano. As células foram estimuladas com extratos de cones de guta-percha convencional, de cones de guta-percha contendo biocerâmica e de cones de polímero em diluição seriada para teste de viabilidade celular por meio do método MTT (Brometo de Difeniltetrazólio 3-(4,5-Dimetiltiazol-2-yl) após 72 h. Em seguida, a diluição de 1/5 foi empregada para estimulação das células por 72 h para detecção da expressão gênica de colágeno tipo I e proteína cementária 1 (CEMP-1) por RT-qPCR. Os dados foram estatisticamente analizados por meio de ANOVA sendo considerados significativos valores de p < 0,05. Os resultados observados de forma que, em extrato puro em extrato puro 1:1, houve comprometimento da viabilidade celular tanto para o extrato de cone de guta-percha quanto para o extrato do cone Cpoint podendo ser considerados citotóxicos. Nas outras diluições não houve diferença significativa neste parâmetro. Em relação à expressão gênica de colágeno, não foram observadas diferenças significativas quando da presença dos extratos. Para CEMP-1, significativa indução da expressão gênica foi observada para o cone de guta-percha. Conclui-se, através da análise dos resultados, que o cone de guta-percha e o cone de polímero são os mais citotóxicos em extrato puro, porém a guta-percha foi o único material que induziu uma expressão significativa de CEMP-1 que auxilia no reparo tecidual. O Col1 não foi induzido em nenhuma das amostras, porém também não foi inibido que indica que nenhum dos 3 tipos de cone interfere no reparo tecidual.Root canal obturation is a fundamental step for successful endodontic treatment. It is desirable that the materials employed at this stage did not adversely interfere with tissue repair but rather stimulate the regeneration of the periapical tissues. Recently, gutta-percha points combined with bioceramic materials were developed for this purpose. Thus, the objective of this study was to investigate the cytotoxic and biomodulatory potential of conventional gutta-percha points, gutta-percha points containing bioceramics and polymer points on periodontal ligament cells in vitro. Culture of periodontal ligament fibroblasts was established from one human third molar. The cells were stimulated with extracts of cones of conventional gutta-percha points, gutta-percha containing bioceramics and polymer points in serial dilution for cell viability test using the MTT assay [Diphenyltetrazolium Bromide 3- (4,5)]. Next, the 1/5 dilution was used to stimulate the cells for 72 h to detect the gene expression of type I collagen and cement protein 1 (CEMP-1) by RT-qPCR. Data were statistically analyzed by means of ANOVA being considered significant values of p <0.05. The results observed was that in a pure 1: 1 extract, there was impairment of cell viability for both the guta-percha cone extract and the Cpoint cone extract and could be considered cytotoxic. At the other dilutions, no significant difference on this parameter was observed. Regarding the gene expression of collagen, no significant differences were observed at the presence of extracts. For CEMP-1, significant induction of gene expression was observed for gutta-percha points. In conclusion, the analysis of the results showed that the gutta-percha and polymer points are the most cytotoxic at pure extract, however gutta-percha was the only material that induced a significant expression of CEMP-1 which assists the tissue repair. Col1 was not induced in any of the samples but was also not inhibited indicating that none of the 3 cone types interfere in tissue repair.
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Sipert, Carla Renata. "Estudo in vitro da produção de quimiocinas e pró-colágeno I por fibroblastos de gengiva, ligamento periodontal e polpa dental humanos." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/25/25142/tde-18012012-094959/.
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Fibroblastos são as células mais numerosas encontradas nos tecidos orais como gengiva, ligamento periodontal e polpa dental. Além de exercerem função estrutural, estas células também desempenham papel importante na resposta imune destes tecidos através do reconhecimento de antígenos e produção de mediadores inflamatórios e citocinas. Evidências apontam ainda para o fato de que fibroblastos não constituem um grupo único de células. Sendo assim, os objetivos deste estudo foram: (I) avaliar a produção diferencial de fibroblastos de gengiva, ligamento periodontal e polpa dental de dentes permanentes e decíduos quanto à produção das quimiocinas CCL3 e CXCL12; (II) avaliar a produção de pró-colágeno I pelas células de polpa e (III) avaliar a expressão diferencial dos fibroblastos quanto a microRNAs. Dentes recentemente extraídos (terceiros molares hígidos) e fragmentos de gengivas saudáveis de três pacientes adultos foram obtidos no Laboratório de Farmacologia e Fisiologia Clínica da Faculdade de Odontologia de Bauru. Caninos decíduos de dois pacientes com indicação para extração por motivos ortodônticos foram obtidos na Clínica de Odontopediatria da mesma unidade. Culturas primárias de fibroblastos de gengiva (n=3), ligamento periodontal (n=3) e polpa de dente permanente (n=3) e polpa dental de dente decíduo (n=2) foram estabelecidas a partir de tecidos humanos por meio de técnica de explant. Após a quarta passagem, a produção de CCL3 e de CXCL12 foi avaliada após estímulo com concentrações crescentes (0 10 µg/mL) de ácido lipoteicóico de Enterococcus faecalis (EfLTA), lipopolissacarídeo de Porphyromonas gingivalis (PgLPS) ou LPS de Escherichia coli (EcLPS) por ELISA após 1, 6 e 24 h. O RNAm para as quimiocinas no grupo estimulado com EcLPS por 24 h foi avaliado por transcrição reversa seguida de reação em cadeia da polimerase quantitativa. A produção de pró-colágeno I por células de polpa estimuladas com EfLTA e PgLPS foi avaliada por imunofluorescência. O perfil de expressão de microRNAs foi investigado por ensaio de microarranjo. A produção de CCL3 foi aumentada (p< 0,05) pelos antígenos empregados, porém de maneira mais evidente para EcLPS em células de gengiva. A quimiocina CXCL12 foi detectada em níveis basais em todos os grupos de células, porém em maiores quantidades em fibroblastos de gengiva seguidos pelos de ligamento periodontal. A adição dos antígenos diminuiu a produção de CXCL12 de maneira distinta entre células e entre antígenos (p< 0,05). Fibroblastos de polpa decídua não apresentaram qualquer alteração na produção desta quimiocina pelos antígenos (p> 0,05). No período experimental de 24 h, a expressão do RNAm para CXCL12 não foi alterada enquanto a de CCL3 não foi detectada. A produção de pró-colágeno I se mostrou aumentada (p< 0,05) na presença do desafio antigênico em células de polpa com exceção para fibroblastos de polpa permanente que apresentaram diminuição na produção desta proteína quando estimulados com EfLTA. Em condições basais, fibroblastos do mesmo doador apresentaram perfil distinto de expressão de microRNAs envolvidos com a produção das proteínas-alvo deste estudo. A expressão de imunomiRs por EcLPS também se mostrou modificada de maneira distinta entre os fibroblastos, em especial os de ligamento periodontal. Com base nestes resultados, pode-se concluir que fibroblastos de diferentes tecidos orais apresentam comportamento diferencial frente a antígenos bacterianos comumente relacionados a patologias que afetam a cavidade oral.Fibroblasts are the dominant cells within oral tissues such as gingiva, periodontal ligament and dental pulp. Besides the architectural maintenance of the connective tissues, fibroblasts are also involved in connective tissue immune response through antigen recognition and production of inflammatory mediators and cytokines. Recent studies also demonstrated that fibroblasts do not constitute a unique group of cells. Taken this togeter, the objectives of the present study were: (I) to evaluate the production of the chemokines CCL3 and CXCL12 by human gingival, periodontal ligament as well as permanent and deciduous dental pulp fibroblasts; (II) to evaluate the production of procollagen I by dental pulp fibroblasts and (III) to evaluate the differential pattern of expression of microRNAs by the oral fibroblasts. Recently extracted teeth (non-carious third molars) and fragments of healthy gingiva from three adults were obtained at the Laboratory for Clinical Pharmacology and Physiology at Dental School of Bauru. Deciduous canines from two patients with orthodontic indication for extraction were obtained at Pediatrics Clinics of Dental School of Bauru. Primary cultures of fibroblasts from gingiva (n=3), periodontal ligament (n=3) as well as permanent pulp (n=3) and deciduous pulp (n=2) were established through an explant technique. After the fourth passage, fibroblasts were challenged with increasing concentrations (0 10 µg/mL) of Enterococcus faecalis lipoteichoic acid (EfLTA), Porphyromonas gingivalis lipopolysaccharide (PgLPS) or Escherichia coli LPS (EcLPS) for 1, 6 and 24 h. The chemokines were assessed through ELISA while the mRNA for CCL3 and CXCL12 (EcLPS at 24 h) were assessed through reverse transcription followed by quantitative polymerase chain reaction. The expression of microRNAs was screened through a microarray assay. The production of CCL3 on cell supernatants was detected in all cellular groups, with higher amounts at gingival fibroblasts. EcLPS induced more important chemokine differences compared to the other antigens. CXCL12 basal levels were higher for gingival fibroblasts followed by periodontal ligament ones, but also detected in dental pulp fibroblasts. The production of this chemokine was decreased by stimulation in a different fashion for each antigen and cell type. Deciduous pulp fibroblasts did not display any differences in CXCL12 synthesis even in the presence of the microbial challenge. No differences were detected at mRNA level for CXCL12, while no expression for CCL3 could be detected at 24 h. Increased production of procollagen type I was observed for dental pulp cells in general, with the only exception for permanent pulp cells which displayed decreased production of the protein with EfLTA. Microarray analysis showed differential expression pattern of microRNAs comparing unstimulated cells from the same donnor. EcLPS was able to alter immunomiRs expression in some of the cellular groups, in particular periodontal ligament fibroblasts. In conclusion, our results showed that fibroblasts from distinct oral tissues display differential behavior against bacterial antigens commonly related to the diseases that affect the oral cavity.
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Valdivia, Maria Alejandra Medina. "Cultura e caracterização de células da granulação óssea in vitro: efeitos proliferativos estimulados por diferentes biomateriais." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/25/25146/tde-03092013-162521/.
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O objetivo deste estudo foi estabelecer cultura primária de células derivadas da granulação óssea (GO) de seres humanos para determinar seu padrão de crescimento in vitro e determinar os efeitos biológicos de três membranas reabsorvíveis feitas de colágeno (BioGide®, GenDerm®, CollaTape®) em culturas de fibroblastos gengivais humanos (FGH) e células da granulação óssea (GO). Foram coletadas amostras de tecido ósseo presente no alvéolo de cicatrização de dois pacientes adultos saudáveis sistemicamente com indicação de cirurgia periodontal regenerativa pela técnica do enxerto ósseo em neoformação. Imediatamente após a coleta, as amostras foram transportadas ao laboratório de cultura de células para estabelecimento da cultura primária. As células foram cultivadas em atmosfera úmida, contendo 5% CO2 a 37oC. A curva de crescimento das células foi determinada por meio de contagem de células viáveis. Após a caracterização da curva de crescimento, foram realizadas a caracterização da amostra por meio de determinação da atividade de fosfatase alcalina e de mineralização. Posteriormente, os efeitos de três diferentes tipos de membranas colágenas sobre a proliferação de células GO e FGH foram investigados por meio do teste MTT. As amostras foram divididas em oito grupos: (1) células FGH em meio DMEM (C-FGH); (2) células FGH em meio DMEM condicionado com membrana GenDerm (GD-FGH); (3) células FGH em meio DMEM condicionado com membrana BioGide (BG-FGH); (4) células FGH em meio DMEM condicionado com membrana ColaTape (CT-FGH); (5) células GO em meio DMEM (C-GO); (6) células GO em meio DMEM condicionado com membrana GenDerm (GD-GO); (7) células GO em meio DMEM condicionado com membrana BioGide (BG-GO); (8) células GO em meio DMEM condicionado com membrana CollaTape (CT-GO). O teste de proliferação celular mostrou que houve aumento significativo (p< 0.05; ANOVA para medidas repetidas) do número de células vitais presentes na cultura nos dias 3 (90,8%), 5 (132,50%), 7 (137,50%) e 10 (227,50%) em relação ao controle (dia 0). Foram observadas atividade de fosfatase alcalina e de mineralização in vitro. Houve aumento do número de células FGH e GO viáveis em todos os grupos (p< 0.05; ANOVA para medidas repetidas). Houve maior efeito proliferativo nas células FGH e GO nos grupos GD e CT, com diferenças estatisticamente significantes entre os grupos (p< 0.05; Mann Whitney) apenas no período de 96 horas. Esses achados sugerem que as células GO apresentam alta atividade de proliferação e síntese, sendo compatíveis com células de linhagem osteoblástica. Duas membranas colágenas testadas exerceram maior ação proliferativa tardia sobre osteoblastos, indicando sua eficácia na regeneração dos tecidos periodontais.The aim of this study was to establish primary culture of cells derived from human bone granulation tissue (GO) in order to determine its growth pattern in vitro and the biological effects of three absorbable collagen membranes (BioGide®, GenDerm®, CollaTape®) in human gingival fibroblasts (FGH) and human bone granulation (GO) cell cultures. Samples of bone tissue present at healing sockets of two systemically healthy adults with indication of periodontal regenerative therapy by the newly forming bone were collected. Immediately after, samples were transported to the laboratory of cell culture to the establishment of primary cultures. Cells were cultivated in humid atmosphere with 95% CO2 at 37oC. Cells growth pattern were determined by counting of viable cells. After characterization of growth pattern, samples were characterized according to alkaline phosphatase activity and mineralization detected by alizarin red. Afterwards, the effects of three different types of collagen membranes on GO and FGH cells were investigated by MTT test. Samples were divided into eight groups: (1) FGH cells in DMEM (C-FGH); (2) FGH in DMEM conditioned by GenDerm® membrane (GD-FGH); (3) FGH in DMEM conditioned with BioGide® (BG-FGH); (4) FGH in DMEM conditioned by CollaTape® (CT-FGH); (5) GO cells in DMEM (C-GO); (6) GO cells in DMEM conditioned by GenDerm® (GD-GO); (7) GO cells in DMEM conditioned by BioGide® (BG-GO); (8) GO cells in DMEM conditioned by CollaTape® (CT-GO). Cell proliferation test showed a significant (p< 0.05; ANOVA for repeated measures) increase in the number of vital cells present in the culture at days 3 (90.8%), 5 (132.50%), 7 (137.50%) and 10 (227.50%) compared to control (dia 0). It was observed alkaline phosphatase activity and mineralization in vitro. There was an increase in the number of FGH and GO viable cells at all groups (p< 0.05; ANOVA for repeated measures). Greater proliferative effect at FGH and GO cells at GD and CT groups, with significant differences between groups (p< 0.05; Mann Whitney) only at 96 hs. Two of the collagen membranes tested exerted greater late proliferative effects on osteoblasts, suggesting its efficacy in the regeneration of periodontal tissues.
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Azevedo, Fabiola Pontes. "Avaliação comparativa do comportamento adaptativo de fibroblastos humanos cultivados de mucosa palatina não marginal e de enxerto gengival em área marginal." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/25/25146/tde-05062013-093746/.
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Enxertos gengivais livres são importantes para garantir condições necessárias para o estabelecimento da homeostasia do periodonto de proteção. O processo de inflamação não ocorre por igual em todos os tecidos conjuntivos do organismo e os fibroblastos têm a capacidade de reagir a estímulos agressivos por meio de liberação de diversas citocinas, que desempenham importante função na formação do infiltrado inflamatório. Até o presente trabalho, não há relatos na literatura acerca da comparação do comportamento dos fibroblastos que compõem a mucosa palatina não marginal e dos fibroblastos provenientes de enxerto gengival livre (EGL) marginal em resistir aos estímulos agressores que ocorrem na doença periodontal. Dessa forma, a proposta do presente trabalho foi investigar se os fibroblastos da mucosa palatina não marginal mudariam seu perfil de secreção de citocinas quando enxertados na margem gengival. Foram coletadas biópsias da mucosa palatina no momento da cirurgia de EGL (período inicial) e após 4 meses (período final) no momento da cirurgia para recobrimento radicular. Os fibroblastos foram cultivados e estimulados com LPS de Porphyromonas gingivalis (Pg) e de Escherichia coli (Ec) por 24h e 48h para avaliação comparativa da expressão de citocinas e mediadores do reparo tecidual, como: IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF e CXCL16. As citocinas foram quantificadas no sobrenadante das células por meio de ensaio imunoenzimático (ELISA). Para a citocina IL-6, os fibroblastos da mucosa palatina não marginal mantiveram o mesmo perfil de secreção quando enxertados na área gengival marginal; para MIP-1α a secreção se mostrou aumentada de forma estatisticamente significativa pelos fibroblastos obtidos do enxerto gengival marginal após 48h de estímulo por Pg em comparação com os fibroblastos da área palatina não marginal; a secreção de IL-8 pelos fibroblastos da mucosa palatina não marginal foi maior em resposta ao desafio por LPS de Pg e os fibroblastos obtidos do enxerto gengival marginal exibiram secreção até mesmo sem o estímulo de LPS; apenas os fibroblastos do enxerto gengival marginal apresentaram secreção de TGF-β, mesmo na ausência de estímulo por LPS; a secreção de VEGF e CXCL16 não foi detectada pelos fibroblastos analisados. Conclui-se que os fibroblastos provenientes de uma mucosa palatina não marginal parecem se adaptar às condições locais quando enxertados na área gengival marginal, oferecendo evidência de sua participação efetiva na produção de mediadores inflamatórios importantes para o processo de homeostasia do periodonto marginal.Free gingival grafts are important to ensure conditions for the establishment of homeostasis of the periodontal soft tissues. The process of inflammation does not occur the same way in all connective tissues and fibroblasts have the ability to respond to aggressive stimuli through the release of various cytokines, which play an important role in the inflammatory infiltrate formation. In literature, there are no studies comparing the behavior of fibroblasts from palatal mucosa (not marginal) and fibroblasts from marginal free gingival graft (FGG) regarding their resistance towards periodontal disease aggressive stimuli. Thus, the purpose of this study was to investigate whether fibroblasts from the palatal mucosa behave differently when grafted to the gingival margin considering their mechanism of cytokine secretion. Biopsies from the palatal mucosa were collected at the time of FGG surgery (initial period) and after 4 months (final period) when surgery for root coverage was performed. The fibroblasts were cultured and stimulated with LPS of Porphyromonas gingivalis (Pg) and Escherichia coli (Ec) for 24 and 48 hours in order to make a comparative evaluation of cytokines and mediators of tissue repair expression, such as IL-6, IL-8/CXCL8, MIP-1α/CCL3, TGF-β, VEGF and CXCL16. Cytokines were measured in the cell supernatant by enzyme immunoassay (ELISA). For cytokine IL- 6, fibroblasts from palatal mucosa maintained the same secretion pattern when grafted to the gingival margin; for MIP-1α the secretion was significantly increased by fibroblasts from the marginal gingival graft after 48 hours of stimulation with Pg when compared to palatal mucosa fibroblasts; IL-8 secretion by palatal mucosa fibroblasts did not increase in response to Pg LPS challenge and fibroblasts from marginal gingival graft showed secretion even without the stimulus of LPS; only fibroblasts from marginal gingival graft showed secretion of TGF-β, even in the absence of LPS stimulation; VEGF and CXCL16 secretion by fibroblasts was not detected. It was concluded that fibroblasts from palatal mucosa seem to adapt to local conditions when grafted to the gingival margin area, providing evidence of its effective participation in the homeostasis of marginal periodontium through the production of important inflammatory mediators.
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Veronesi, Giovana Fuzeto. "Efeitos do condicionamento com diferentes soluções e tempos de aplicação na descontaminação da superfície radicular, adesão e proliferação de fibroblastos gengivais e de ligamento periodontal: estudo em microscopia eletrônica de varredura." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/25/25146/tde-13072018-095900/.
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O objetivo deste estudo foi investigar in vitro a influência do tratamento de superfícies radiculares na adesão e proliferação de fibroblastos gengivais e de ligamento periodontal humano em fragmentos radiculares de dentes humanos extraídos por razões periodontais. Todos os fragmentos receberam raspagem e alisamento radicular (RAR), e em seguida aleatoriamente divididos em grupos, de acordo com a substância utilizada no tratamento de superfície (n= 15/grupo): ácido fosfórico 37% aplicado por 90s (AF90) ou 180s (AF180) (RAR); EDTA 24% aplicado por 90s (EDTA90) ou 180s (EDTA180); ácido cítrico pH 1.0 a 10% aplicado por 90s (AC90) ou 180s (AC180); ácido cítrico pH 1.0 a 10% associado à tetraciclina 50% por 90s (ACTC90) ou 180s (ACTC180); tetraciclina hidroclorídrica (50mg/ml) aplicada por 90s (TC90) ou 180s (TC180). O grupo controle foi composto por fragmentos tratados por meio de RAR seguido de lavagem em soro fisiológico. Após a realização dos tratamentos, os espécimes (n=3/grupo) foram preparados para análise em MEV com o objetivo de avaliar a descontaminação das superfícies radiculares por meio dos índices de rugosidade superficial (IRS), cálculo residual (ICR), perda de substância dentária (IPSD), presença de restos teciduais (IPRT), remoção de smear layer (IRSL), abertura dos túbulos dentinário (ATD) e smear layer remanescente (SLR) em fotomicrografias em aumentos de 500x e 1000x. Em 6 espécimes de cada grupo, foram plaqueados 104 fibroblastos gengivais (FGH-1), e em outros 6, 104 fibroblastos de ligamento periodontal humano (FLP-1). Após 24h foram fixados 6 espécimes por grupo (n=3/grupo) e após 48h os outros 6 espécimes (n=3/grupo), para análise em microscópio eletrônico de varredura. Para determinar a adesão e proliferação celular, o número de células aderidas à superfície nos dois períodos de avaliação foi determinado em triplicatas por um examinador independente A comparação entre os grupos foi realizada pelo método Kruskal-Wallis complementado pelo teste de Dunn para variáveis não lineares, e por meio de análise de variância múltipla (ANOVA) complementado pelo teste de Tukey para as variáveis lineares. A comparação entre os pares nos períodos de 24 e 48 horas foi realizada por meio do método ANOVA pósteste Sidak para variáveis lineares, e Kruskal Wallis pós-teste Dunn para variáveis não lineares. Foi adotado nível de significância de 5% em todos os testes. Não houve diferença estatisticamente significante para IRS, IPSD, IPRT, IRSL, ATD e SLR. Houve maior quantidade de cálculo residual nos grupos TC90 (3,66 ± 0,57; mediana = 4) e AF180 (3,66 ± 0,57; mediana = 4), enquanto que o grupo AC90 (1,33 ± 0,57; mediana = 1) mostrou quantidade significativamente menor de cálculo residual. Encontrou-se uma adesão significativamente maior de células FGH-1, no grupo EDTA180 (170 ± 77,99) no período de 24 horas, e maior efeito proliferativo (48 horas) no grupo TC90 (172,90 ± 65,38). Para células FLP-1, observou-se uma adesão significativamente maior no grupo ACTC90 (74,67 ± 98,84) no período de 24 horas e maior efeito proliferativo (48 horas) no grupo AC90 (173,8 ± 139,6). A partir dos resultados obtidos, sugere-se que a substância a ser utilizada para o condicionamento das superfícies radiculares seja escolhida de acordo com os objetivos do tratamento periodontal: quando se objetiva a formação de nova inserção conjuntiva o uso do EDTA ou AF por 180s ou da tetraciclina por 90s; para regeneração dos tecidos periodontais, sugere-se o uso de AC por 90s.The aim of this study was to evaluate in vitro the influence of root surface conditioning on adhesion and proliferation of gingival and periodontal ligament fibroblasts on human root fragments of teeth extracted for periodontal reasons. Fragments received scaling and root planning (SRP), and were then randomly allocated into groups according to the substance used for root surface treatment (n= 15/grupo): phosphoric acid 37% applied for 90s (PA90) or 180s (PA180); EDTA 24% applied for 90s (EDTA90) or 180s (EDTA180); 10% citric acid pH 1.0 applied for 90s (CA90) or 180s (CA180); 10% citric acid pH 1.0 associated to tetracycline HCL 50% applied for 90s (CATC90) or 180s (CATC180); tetracycline hydrocloride (50mg/ml) applied for 90s (TC90) or 180s (TC180). Control group was composed by SRP treated root fragments, followed by saline solution washing. After treatment completion, specimens (n=3/grupo) were prepared for scanning electronmicroscopy (SEM) analysis, aiming at evaluation of its surfaces according to the following indexes: superficial roughness (SR); residual calculus (RC); loss of tooth substance (LT); tissue residual (TS), smear layer removal (SLR), dentin tubules opening (DTO) and smear layer residual (SLR) in photomicrographs on 500x and 1000x magnifications. In 6 specimens of each group 104 gingival fibroblasts (HGF-1) were plated; and over another 6 specimens, 104 periodontal ligament fibroblasts (PLF-1). After MEV evaluation, the number of cells adhered to the root surfaces over 24h and 48h were assessed by a calibrated examiner in triplicates. Groups comparison were analyzed through Kruskal-Wallis post-test Dunn for comparisons for non-linear variables, and ANOVA post-test Tuckey for linear variables. Comparison between pairs over 24 and 48 hours was accessed through Kruskal-Wallis post-test Dunn for non-linear variables, and ANOVA post-test Sidak for linear variables. Significance level of 5% was adopted in all tests. There was no statistical difference for SR, LT, TS, SLR, DTO and SLR. Although there was higher amounts of residual calculus on groups TC90 (3,66 ± 0,57; median = 4) and FA180 (3,66 ± 0,57; median = 4) while group CA90 (1,33 ± 0,57; median = 1) showed statistically less residual calculus. A singnificantlly higher HGF-1 cell count was found on EDTA180 (170 ± 77,99) on 24-hour period and a higher proliferative effect (48 hours) on group TTC90 (172,90 ± 65,38). A significantly higher cell adhesion for (PLF-1) was found on group ACTC90 (74,67 ± 98,84) at 24-hour assessment, and higher proliferative effect (48 hours) for AC90 (173,8 ± 139,6). From the data here exposed, it is suggested that the substance election for root surface conditioning should be based on the treatment primary goal: when a new connective tissue adhesion is aimed, EDTA or PA for 180s or TTC for 90s should be chosen; on the other hand, for periodontal regeneration, CA for 90s should be the best option.
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Morandini, Ana Carolina de Faria. "Produção diferencial de pró-colágeno tipo I e citocinas por fibroblastos humanos de ligamento periodontal e de gengiva estimulados por lipopolissacarídeo de Porphyromonas gingivalis." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/25/25135/tde-25052009-142548/.
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O ligamento periodontal e o tecido gengival são formados por tecido conjuntivo frouxo, sendo constituídos por diversas células, dentre as quais os fibroblastos são as mais numerosas. Ao serem submetidas a agressões diversas, estas células respondem com a liberação de substâncias, tais como citocinas e quimiocinas, que participam de maneira ativa no processo inflamatório, e com a produção de componentes da matriz extracelular fundamentais para o reparo, como por exemplo, o colágeno. Assim sendo, este trabalho teve como objetivo: Avaliar e comparar a expressão e a produção de prócolágeno tipo I, IL6, MIP1 e SDF1 por fibroblastos humanos, cultivados de ligamento periodontal e de tecido gengival, estimulados por lipopolissacarídeo (LPS) de Porphyromonas gingivalis. Foram coletados ligamentos periodontais de terceiros molares não irrompidos e biópsias de gengiva de um mesmo indivíduo (n=3). Estes tecidos foram picotados e mantidos em meio de cultura adequado para fibroblastos, que foram utilizados na quarta passagem. Após adesão dos fibroblastos a placas de 24 poços, o meio de cultura contendo 0,1 10 g/mL de LPS de P. gingivalis foi adicionado às placas em duplicata. O sobrenadante e as células foram coletados após 1, 6 e 24 horas e analisados por ELISA e PCR em tempo real, respectivamente. A análise estatística foi realizada por meio do programa GraphPad Prism, aplicandose o teste ANOVA a 1 critério com nível de significância de 5%. A expressão de prócolágeno tipo I mostrouse - ligeiramente diferente entre fibroblastos de ligamento periodontal e de gengiva. A produção de IL6, MIP1 e SDF1 foi significativamente maior em fibroblastos gengivais. A citocina IL6 foi produzida de maneira tempodependente com LPS de P gingivalis, principalmente por fibroblastos gengivais. Para MIP1, os fibroblastos gengivais mostraram maior produção com a menor concentração de estímulo (0,1g/ml). Para SDF1, foi detectada produção constitutiva que foi inibida com o aumento da concentração de LPS ao longo do tempo nestas mesmas células. Já para fibroblastos de ligamento periodontal, não foi observado um padrão homogêneo e linear, apesar de a produção basal de SDF1 também existir, porém em níveis bem mais discretos, como aquele observado para a produção de MIP1. A capacidade dos fibroblastos modificarem o padrão de produção dessas citocinas frente ao estímulo com LPS de P. gingivalis reforça a importância dessas células no contexto da resposta imune do indivíduo frente à doença periodontal.The fibroblast is considered an important cell in periodontitis because it is the predominant cell type in the periodontal connective tissue. When challenged by different agents, fibroblasts respond through the release of substances, such as cytokines and chemokines that participate in an active way in the inflammatory process as well as the production of basic components of the extracellular matrix for repair, like collagen. The aim of this study was to: to evaluate and to compare the expression and production of type I procollagen, IL6, MIP1 and SDF1 by cultured human periodontal ligament and gingival fibroblasts challenged with lipopolyssacharide from Porphyromonas gingivalis. Human periodontal ligament and gingival fibroblasts were cultured from biopsies of the same donor and were used on the fourth passage. After confluence in 24well plates, the culture medium alone (control) or with 0,1 10 ug/mL of LPS from P. gingivalis were added and after 1, 6 and 24 hours, the supernatant and the cells were collected and analysed by ELISA and Real time PCR, respectively. Data were analysed by GraphPad Prism Program (1 way ANOVA test) and a significance level of 5% was adopted. Procollagen type I expression by Real Time PCR differ between periodontal ligament and gingival fibroblasts. In vitro experiments revealed that IL6, MIP 1 and SDF1 production were significantly greater in gingival fibroblasts when compared to periodontal ligament. In addition, IL6 was upregulated in a timedependent manner, mainly by the gingival fibroblasts. On one hand, MIP1 was stimulated with a low concentration (0,1ugml) of LPS by gingival fibroblasts. On the other hand, SDF1 was constitutively secreted by the same cells but its production was inhibited when challenged by a higher concentration of LPS from P gingivalis. In general, periodontal ligament fibroblasts did not show a pattern of production of these cytokines under the challenge with LPS, despite of the basal production of SDF1 in lower levels than gingival cells and the low production of MIP1 over time. The differential ability of the gingival and periodontal ligament fibroblasts to secrete these cytokines emphasizes their crucial role in the inflammatory microenvironment and in the host immune response to periodontal disease.
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Bedran, Telma Blanca Lombardo. "Efeito antimicrobiano e modulador da resposta imune dos peptídeos hBD-3 e LL-37 e dos polifenóis o chá verde e do cranberry /." Araraquara, 2014. http://hdl.handle.net/11449/124090.
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Orientador: Denise Madalena Palomari SpolidórioBanca:Juliana Rico Pires
Banca: Shelon Cristina Souza Pinto
Banca: Luciene Cristina Souza Pinto
Banca: Joni Augusto Cirelli
Resumo:Os peptídeos antimicrobianos como por exemplo a catelicina (LL-37) e as defensinas humanas (hBD-1, hBD-2 e a hBD-3) são considerados antibióticos endógenos com importante papel na prevenção das doenças periodontais, devido a sua capacidade de regulação da resposta imune, sendo que os mesmos podem ser degradados pelos periodontopatógenos. Terapias que aumentem a produção destes peptídeos pelas próprias células do organismo, assim como a associação destes peptídeos com compostos naturais os quais possam agir em sinergismo na regulação da resposta imune, podem ser considerados novas estratégias para o melhor controle das doenças periodontais. Portanto os objetivos deste estudo in vitro foram: i) Avaliar a capacidade do extrato do chá verde (Camellia sinensis) e do seu polifenol, o EGCG, sobre a expressão gênica de hBD-1 e hBD-2 pelas células epiteliais gengivais (B11), sobre a degradação das mesmas frente ao P. gingivalis, ii) Através da utilização do modelo 3D de co-cultura celular, avaliar a capacidade antiinflamatória dos peptídeos hBD-3 e LL-37 quando em associação sobre a produção de citocinas, quimiocinas e fatores de crescimento, iii) Avaliar a capacidade anti-inflamatória da associação do EGCG e do polifenol proveniente do cranberry, o AC-PACs, com o peptídeo LL-37 sobre a produção de citocinas, quimiocinas e fatores de crescimento em modelo de co-cultura celular. As células epiteliais gengivais (B11) foram estimuladas com o extrato do chá verde e com o EGCG na presença e ausência de inibidores específicos. A produção e expressão gênica de hBD-1 e hBD-2 foram quantificados respectivamente pelas técnica de ELISA e qPCR. A capacidade do extrato do chá verde e do EGCG em proteger a degradação de hBDs pelo P. gingivalis foi mensurado através da técnica de ELISA. Foi desenvolvido um modelo em 3D de co-cultura de fibroblastos gengivais embebidos em....(Resumo completo, clicar acesso eletrôni
Abstract: The antimicrobial peptides LL-37, hBD-1, hBD-2 and hBD-3 are considered an endogenous antibiotic, with important role in the prevention of periodontal diseases due to their ability to regulate the immune response. However those peptides could be degraded by periodontal pathogens. Therefore, therapies able to up regulate the secretion of those peptides by human cells, and the association of antimicrobial peptides with natural compounds, which may act in synergism to modulate the immune response, may be a novel approach for effectively controlling periodontal diseases. The aim of this in vitro study were: i) investigate the ability of green tea extract and EGCG to induce hBD-1 and hBD-2 secretion and gene expression by gingival epithelial cells (B11) and to protect hBDs from degradation by P. gingivalis, ii) A 3D co-culture model of gingival epithelial cells and fibroblasts stimulated with A. actinomycetemcomitans LPS (1 μg/ml) were used to investigated the anti-inflammatory properties of the hBD-3, LL-37, ACPACs and EGCG and to determine whether LL-37 acts in synergy with AC-PACs, EGCG and hBD-3. Gingival epithelial cells were stimulated with green tea extract or EGCG in the presence and absence of specific inhibitors. The secretion and gene expression of hBD-1 and hBD-2 was respectively measured by ELISA and qPCR. The ability of green tea extract and EGCG to prevent hBDs degradation by P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA. A 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to A. actinomycetemcomitans LPS stimulation compared to fibroblasts and epithelial cells individually. The 3D co-culture model was stimulated with noncytotoxic concentrations of: i) hBD-3 (10 and 20 μM) ...(Complete abstract electronic access below)
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Cano, Rodrigo das Neves. "Papel da MMP-9 e do TGF-β na expressão de componentes da Matriz Extracelular por fibroblastos gengivais de camundongos normais e diabéticos com doença periodontal in vitro /." Araçatuba, 2019. http://hdl.handle.net/11449/183465.
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Orientador: Sandra Helena Penha de OliveiraResumo: Na presença de Diabetes Mellitus, a doença periodontal (DP) pode acentuar o aparecimento de complicações e aumentar sua prevalência. O objetivo desta pesquisa foi avaliar se o Fator de Crescimento Transformador -β do tipo 1 (TGF-β1, sigla em inglês) e Metaloproteinase de matriz -9 (MMP-9, sigla em ingês) estão modulando a formação de componentes de matriz extracelular em fibroblastos gengivais (FG) de camundongos diabéticos e com DP. Os 24 animais foram separados em 4 grupos, e então foram tratados com estreptozotocina para indução de diabetes e foi realizado indução da DP através de ligadura com fio de seda. Após a eutanásia e coleta da gengiva, FG dos camundongos foram cultivados. A expressão gênica de RNAm e produção de proteína para MMP-9, TGF-β1 e colágeno tipo 1 (Col1a1) foram avaliadas. Eles também foram tratados com inibidores de MMP-9 e TGF-β1, e dexametasona (Dexa) por 24h. FG de animais normais e diabéticos com DP induzida tiveram expressão aumentada de MMP-9 e TGF-β1 em 6 e 24h, diminuindo em 48h. Expressão gênica de Col1a1 foi diminuída em FG com DP em 6, 24 e 48h. Inibidor de MMP-9 (MMP-9i) bloqueou a expressão gênica de MMP-9 e de TGF-β1 em FG normais e diabéticos em 24h. A expressão de col1a1 foi inibida pelo MMP-9i somente em FG normais, mas não em diabéticos. A expressão de receptor de TGF-β do tipo 1 (TGF-β R1) foi aumentada em FG normais e diabéticos, porém MMP-9i diminuiu esta expressão somente em FG diabéticos. Inibidor de TGF-β (SB431542) e Dexa interro... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In presence of Diabetes Mellitus, periodontal disease (PD) can accentuate complication appearance and increase prevalence. The aim of this study was to evaluate whether Transforming Growth factor-β 1 (TGF-β1) and Matrix Metaloproteinase-9 (MMP-9) are modulating the extracellular matrix components in gingival fibroblasts (GF) from diabetic mice (D) with PD. 24 animals were divided in 4 groups then they were treated with streptozotocin to diabetes induction and DP was inducted through a ligature with silk thread. After euthanasia and gingiva harvest, mice GF were cultured. MMP-9, TGF- β1, and type 1 Collagen (col1a1) mRNA expression and protein production were evaluated. They were also treated with MMP-9 and TGF-β inhibitors, and dexamethasone (Dexa) for 24h. Normal and diabetic GF from PD-induced mice had increased MMP-9 and TGF-β1 expression at 6 and 24 h decreasing at 48 h. Col1a1 gene expression was decreased in normal GF with PD at 6, 24 and 48 h. In diabetic GF col1a1 expression was inhibited at 6, 24 and 48 h. MMP-9 inhibitor (MMP-9i) blocked the MMP-9 expression, and TGF-β1 gene expression in normal and diabetic GF at 24 h. The col1a1 gene expression was inhibited by MMP-9i only in normal GF but not in diabetic. TGF-β R1 was increased in normal and diabetic GF with PD, but MMP-9i decreased this expression only in diabetic GF. TGF-β inhibitor (SB431542) and Dexa abrogated MMP-9, TGF-β1 and col1a1 expression in normal and diabetic GF with PD at 24 h. TGF-β R1 was also inh... (Complete abstract click electronic access below)
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Crowley, Thomas. "Innate immune memory in fibroblasts." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8060/.
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The innate immune system is a generic response to infection or injury. Evidence shows the innate response has immunological memory capable of altering subsequent responses to stimuli. Fibroblasts are ubiquitous stromal cells capable of responding to inflammatory triggers, and of orchestrating endothelial cell and leukocyte behaviour during inflammation. Repeated challenge with cytokines (such as tumour necrosis factor (TNF) a) induced an augmented second response to stimulation. Fibroblasts from multiple anatomical locales significantly increased cytokine secretion upon second challenge with TNFa. The precise mediators augmented depended on fibroblast site of origin. Depending on site, memory was inherent, or only present in fibroblasts from chronically-inflamed tissue. This suggests a phenomenon intrinsic to some sites but pathological in others. The secreted mediators from the fibroblast initial or memory responses exerted differing effects on leukocytes, dependent upon fibroblast site of origin. Finally, examination of intracellular signalling showed the augmented response was at least partly due to prolonged activity of nuclear factor (NF) KB during the memory response. Innate immune memory exists in fibroblasts from multiple tissues, but may be pathologically acquired in some. The altered response to second challenge may represent a fibroblast mechanism for altering the recruitment and behaviour of the inflammatory infiltrate.
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Benanti, Jennifer Ann. "Regulation of cellular senescence in human fibroblasts /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/5050.
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Antczak, Michael Richard. "Growth factor- and oncogene-induced transformation in chicken embryo fibroblasts and normal diploid human fibroblasts." Case Western Reserve University School of Graduate Studies / OhioLINK, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=case1057173851.
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Damante, Carla Andreotti. "Efeito da terapia com laser em baixa intensidade (LILT) na expressão de fatores de crescimento da família FGF por fibroblastos gengivais humanos." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/23/23146/tde-10082007-125623/.
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A terapia com laser em baixa intensidade, conhecida como Low-intensity laser therapy (LILT), tem sido alvo de pesquisas que buscam esclarecer os mecanismos pelos quais o laser age na cicatrização de feridas. Este processo é dependente da transmissão de sinais entre epitélio e conjuntivo, os quais influenciam a proliferação e a migração celular, onde participam fatores de crescimento. O objetivo desta pesquisa foi verificar os efeitos da radiação com lasers vermelho e infravermelho em baixa intensidade na produção de fatores de crescimento por fibroblastos in vitro. Foi obtida uma cultura primária de fibroblastos gengivais humanos (linhagem FGH). As células foram cultivadas em placas de 96 poços (5 x 103 células /poço), em estado de quiescência (meio de cultura suplementado com 1% de soro fetal bovino) e irradiadas com lasers de diodo (AlGaAs - 660nm, e AlGaInP - 780nm). O laser em modo contínuo, foi aplicado em contato e na forma pontual. A potência utilizada foi de 40mW numa área de 0,042cm2 e com densidades de energia de 2, 3, 4, 5 e 6J/cm² com respectivos tempos de aplicação de 2, 3, 4, 5 e 6s. As culturas foram irradiadas duas vezes com 6h de intervalo. Os grupos controle positivo e negativo foram cultivados com 10% e 1% de soro fetal bovino, respectivamente e não receberam irradiação. A produção do fator de crescimento de queratinócitos (KGF) e do fator de crescimento de fibroblastos básico (bFGF) foi analisada e quantificada por ensaio imunoenzimático (método ELISA) no meio condicionado pelas células. Os dados foram comparados estatisticamente pela análise de variância e teste de Kruskal-Wallis complementados pelos testes de Tukey e Dunn, respectivamente (p<0,05). Culturas de todos os grupos produziram KGF e bFGF. Controles positivos produziram maior quantidade de KGF e bFGF que controles negativos. A produção de KGF foi similar em todos os grupos experimentais. A produção de bFGF foi significativamente maior nos grupos tratados com o laser infravermelho nas densidades de energia de 3J/cm2 e 5J/cm2 quando comparados com os controles. Baseados nas condições deste estudo, concluiu-se que a LILT não influencia a produção de KGF, porém, influencia positivamente a produção de bFGF de maneira dependente do comprimento de onda e da densidade de energia empregados. O infravermelho, nas densidades de energia de 3J/cm2 e 5J/cm2, é capaz de aumentar a produção deste fator de crescimento. Portanto, a utilização da LILT nestes parâmetros poderá constituir papel relevante na cicatrização de feridas.Low-intensity laser therapy (LILT) has been the subject of researches to understand the mechanisms by which the laser acts stimulating wound healing. This process is dependent of a signaling transfer between epithelium and connective tissue and it influences cellular proliferation and migration. Growth factors are directly involved in these mechanisms. The aim of this study was to analyze the effects of red and infrared low-level laser on production of fibroblast growth factors by human gingival fibroblasts in vitro. The cells were obtained by primary culture (FGH cell line). They were grown in 96 wells plates (5x10³ cells / well) in a quiescence environment (1% fetal bovine serum) and then they were irradiated with a diode laser (GaAlAs - 660nm, and InGaAlP - 780nm). The continuous laser was applied in contact and in a punctual mode. The power was 40mW, the area of irradiation was 0,042cm2, the energy densities were 2, 3, 4, 5 and 6J/cm² with 2, 3, 4, 5 and 6s of time application, respectively. The cultures were irradiated twice with 6 h-interval. The positive and negative control groups were grown in 10% and 1% of bovine fetal serum, respectively and didn?t receive any irradiation. The production of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) was analyzed and quantified by immunoenzymatic test (ELISA) in the conditioned medium. The data were compared statistically by analysis of variance and Kruskal-Wallis, complemented by Tukey?s and Dunn?s tests, respectively (p<0,05). Positive controls produced more KGF and bFGF than negative controls. The KGF production was similar in all experimental groups. The bFGF production was significantly higher in groups treated with infrared laser with energy densities of 3J/cm² and 5J/cm² when compared to controls. Based on these study conditions, authors concluded that LILT doesn?t influence KGF production although it positively influences bFGF production in a dependent manner from wavelength and energy density. Infrared laser with energy densities of 3J/cm² and 5J/cm² is capable of improving the production of this growth factor. Therefore the LILT in these parameters can constitute a relevant role on wound healing.
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Freiin, von Feilitzsch Margarete. "Einfluss von modifizierter extrazellulärer Matrix auf die Proteinexpression von Fibroblasten." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-172391.
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Der humanen dermalen Wundheilung liegt ein komplexes Zusammenspiel verschiedener Faktoren zugrunde. Die Bedeutung dieses fein regulierten Gleichgewichts wird deutlich, wenn es durch Fehlregulationen oder Störungen zu chronischen Wundheilungsstörungen oder lokaler Fibrose mit überschießender Narbenbildung kommt. Eine der möglichen Methoden zur Prävention und Behandlung ist die Deckung der Wunde mit einem Hautersatz. Dabei werden zunehmend sogenannte Biomaterialien aus natürlichen Substanzen mit hoher Biokompatibilität und der Möglichkeit zur Interaktion mit dem nativen Gewebe verwendet. In Studien wurde gezeigt, dass vor allem sulfatierte Glykosaminoglykan-Derivate durch die Interaktion ihrer negativ geladenen Sulfatgruppen mit Zytokinen, Wachstumsfaktoren und dermalen Zellen einen positiven Einfluss auf den Wundheilungsprozess haben können. In der vorliegenden Arbeit wurden daher kollagenbasierte artifizielle extrazelluläre Matrizes mit unsulfatierter oder sulfatierter Hyaluronsäure hinsichtlich ihres Einflusses auf humane dermale Fibroblasten als Komponenten der Wundheilung untersucht. Dermale Fibroblasten spielen im Ablauf der Wundheilung eine tragende Rolle und interagieren eng mit der umgebenden Matrix. Anhand ihrer Proteinexpression lassen sich Rückschlüsse auf wichtige Funktionen wie Adhäsion, Proliferation, Differenzierung und Matrixsynthese ziehen. In den durchgeführten Experimenten zeigte sich, dass sulfatierte Matrix in der Kultur mit dermalen Fibroblasten kein entzündliches Milieu förderte. Die Proliferation, Differenzierung und Migration der Fibroblasten schienen gesteigert, während sich die Matrix-Synthese und ihr Remodeling weder pathologisch gehemmt noch überschießend zeigten. Daher wäre die weitere Untersuchung dieses Biomaterials ein vielversprechender Ansatz, um langfristig dem Risiko von Wundheilungsstörungen wie chronischen Wunden oder fibroproliferativen Wundheilungsstörungen effektiv entgegenzuwirken.
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Ribeiro, Ana Paula Dias [UNESP]. "Efeito citotóxico da Terapia Fotodinâmica associando Photogem e LED azul e vermelho em cultura de células normais." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/98035.
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ribeiro_apd_me_arafo.pdf: 10016445 bytes, checksum: 54f364c1faa5066e22a82916fe0e1ac2 (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Para considerar a Terapia Fotodinâmica (PDT) como tratamento clínico da estomatite protética, é necessário conhecer tanto o potencial antifúngico, como efeito citotóxico desta terapia sobre células normais do indivíduo. Assim, o objetivo desse estudo in vitro foi avaliar a citotoxicidade da PDT antifúngica com o fotossensibilizador Photogem® associado ao LED azul e ao LED vermelho em cultura de fibroblastos L929 e células odontoblastóides MDPC-23. As células foram cultivadas (30.000 células/cm2) em placas de 24 compartimentos por 48 horas e ambos os tipos celulares foram incubados com Photogem® (0, 10, 25, 50, 100 ou 150 mg/L) e irradiados ou não pelo LED azul (460 ± 3 nm; 25,5 ou 37,5 J/cm2; 22 mW/cm2) ou LED vermelho (630 ± 3 nm; 70 ou 100 J/cm2; 25 mW/cm2) . O metabolismo celular foi determinado 0, 12 e 24 horas após a PDT utilizando o teste do metiltetrazolium (MTT), e a morfologia celular avaliada pela microscopia eletrônica de varredura (MEV). A técnica de citometria de fluxo foi utilizada para avaliar o tipo de morte celular (necrose ou apoptose) assim como estimar os níveis intracelulares das espécies reativas de oxigênio (EROs). Observou-se redução do metabolismo celular estatisticamente significante para todas as concentrações do Photogem® quando irradiadas em qualquer dose de luz, sendo essa redução de 90 a 97% tanto para as células L929 quanto MDPC-23 (ANOVA and Dunnet’s post hoc tests; p<0.05). Essa redução da atividade mitocondrial não foi dependente da concentração do fotossensibilizador e nem da dose de luz empregada. Também, foi demonstrado que a atividade mitocondrial das células submetidas a PDT não foi recuperada após 12 ou 24 horas, caracterizando um dano irreversível. A presença do Photogem® e da luz isoladamente não alterou estatisticamente a atividade mitocondrial de ambas as linhagens. As células submetidas...
In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its antifungal potential and its cytotoxicity effect on normal cells. Therefore, the purpose of this in vitro study was to evaluate the cytotoxicity of PDT with Photogem® associated to blue and red LED on L929 and MDPC-23 cell cultures. The cells (30.000 cells/cm2) were seeded in 24-well plates for 48 hours, incubated with Photogem® (10, 25, 50, 100 or 150 mg/L) and were irradiated or not with a blue LED source (460 ± 3 nm; 25.5 or 37.5 J/cm2; 22 mW/cm2) or with a red LED source (630 ± 3 nm; 75 or 100 J/cm2; 22 mW/cm2). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet’s post hoc tests; p<0.05) and cell morphology was examined by scanning electron microscopy. Flow cytometry was employed to analyze the type of PDT-induced cell death (necrosis or apoptosis) as well as to estimate intracellular production of reactive oxygen species (ROS). There was a statistically significant decrease of mitochondrial activity for all Photogem® concentrations associated to blue or red LED regardless irradiation time; this reduction ranged from 90 to 97% for both cell lines. This reduction, however, was not dependent on the photosensitizer concentration. It was also demonstrated that the mitochondrial activity of the cells submitted to PDT was not recovered after 12 or 24 hours, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death when in contact with the photosensitizer. The presence of Photogem® and exposure to blue and red LED increased the intracellular levels of ROS in both L929 and MDPC- 23 cells in a dose-dependent manner. The association of Photogem® and blue LED caused severe toxic effects on normal cell culture... (Complete abstract click electronic access below)
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Santos, Caroline de Moura Martins Lobo dos. "Fibroblastos gengivais humanos em co-cultura com Aggregatibacter actinomycetemcomitans lisogênico induzem a liberação de fago." Universidade do Estado do Rio de Janeiro, 2011. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=3791.
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A relação entre bacteriófagos e virulência bacteriana é um modelo muito intrigante e pouco estudado na patogênese periodontal, uma vez que um patógeno periodontal pode ser lisogênico. O objetivo do nosso estudo é determinar a capacidade de fibroblastos gengivais humanos de induzir as cepas lisogênicas de Aggregatibacter actinomycetemcomitans. Dois experimentos foram realizados seguidos da titulação de fago. Os experimentos consistiram da co-cultura com fibroblastos gengivais humanos e três cepas de Aa [Aa29524, Aa2112, Aa29524(Ø2112)], não lisogênica, lisogênica e lisogênica induzida em laboratório, respectivamente. Em três momentos distintos (no experimento 1: 0, 2 e 4 horas; e no experimento 2: 2, 4 e 6 horas), o sobrenadante da co-cultura foi filtrado e cultivado overnight com a bactéria indicadora (Aa29524) e analisado para a capacidade de lisar a célula indicadora. Em ambos os experimentos, o sobrenadante da co-cultura de fibroblastos gengivais humanos com Aa lisogênico e Aa lisogênico induzido em laboratório, ao ser cultivado com a bactéria indicadora, promoveu lise da mesma, resultando no aumento da produção de fago. Pode-se concluir que, nesse estudo os fibroblastos gengivais humanos foram capazes de induzir cepas lisogênicas de Aggregatibacter actinomycetemcomitans.The relationship between bacteriophages and bacterial virulence is a very intriguing, but rarely studied model in periodontal pathogenesis, as a periodontal pathogens can be lysogenic. The aim of our study is to determine the ability of human gingival fibroblasts to induce lysogenic strains of Aggregatibacter actinomycetemcomitans. Two experiments were performed followed by titration of phage. The experiments consisted of co-culture with human gingival fibroblasts and three strains of Aa [Aa29524, Aa2112, Aa29524 (Ø2112)], not lysogenic, lysogenic and lysogenic induced in the laboratory, respectively. In three different times (in experiment 1: 0, 2 and 4 hours, and in experiment 2: 2, 4 and 6 hours), the co-culture supernatant was filtered and cultured overnight with the indicator strain (Aa29524) and analyzed for ability to lyse the cell indicator. In both experiments, the supernatant of the co-culture of human gingival fibroblasts with Aa lysogenic and Aa lysogenic induced in the laboratory to be cultured with the indicator bacteria, caused lysis of the same, resulting an increased phage production. It can be concluded that in this study, human gingival fibroblasts were able to induce lysogenic strains of Aggregatibacter actinomycetemcomitans.
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Santana, Luís Carlos Leal. "Impacto de andrógenos sobre a proliferação e atividade de fibroblastos e células epiteliais em cultura celular /." Araraquara, 2016. http://hdl.handle.net/11449/138766.
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Orientador: Luis Carlos SpolidórioResumo: Hormônios esteroides sexuais participam de diversos eventos celulares e moleculares, e exercem influência sobre o epitélio e tecido conjuntivo do periodonto. A testosterona (T), principal hormônio androgênico, pode ser convertida em estradiol (E2) pela ação da enzima aromatase, ou em di-hidrotestosterona (DHT) pela ação da enzima 5α-redutase. Para elucidar o impacto de andrógenos sobre as células que compõem os tecidos conjuntivo e epitelial, fibroblastos e queratinócitos foram avaliados em relação aos efeitos de diferentes concentrações de T e DHT, além da exposição ao anastrozol (ANA), flutamida (FLU), fulvestranto (FUL), e às associações farmacológicas T+ANA, T+FLU e T+FUL. Os resultados do presente estudo indicaram que, de modo geral, hormônios esteroides androgênicos exercem efeitos opostos sobre eventos celulares de fibroblastos gengivais humano e células epiteliais HaCaT em cultura celular. Enquanto a T e a DHT agem promovendo o aumento da proliferação e atividade de fibroblastos, a exposição de células HaCaT a estes mesmos andrógenos resulta em inibição ou exiguidade do crescimento celular, atividade metabólica ou a capacidade de repovoamento da área de arranhão in vitro. Além disso, o tratamento farmacológico com ANA, FLU, FUL, e suas respectivas associações à T, parece influenciar eventos celulares de fibroblastos gengivais humano e células epiteliais HaCaT in vitro.
Abstract: Sex steroid hormones take part in different cellular and molecular process and exert their functions on the epithelium and connective tissue of the periodontium. Testosterone (T), the main androgenic hormone can be converted to estradiol (E2) through the aromatase enzyme action, or into dihydrotestosterone (DHT) by 5α-reductase activity. To elucidate the impact of androgens on the cells that constitute the connective and epithelial tissues, fibroblasts and keratinocytes were evaluated under the effects of different concentrations of T and DHT, besides to be both exposed to anastrozole (ANA), flutamide (FLU), fulvestrant (FUL), and the pharmacological associations T+ANA, T+FLU and T+FUL. The results of this study indicated that, in general, androgenic steroid hormones exert opposite effects on cellular events of human gingival fibroblasts and epithelial cells. While androgens act stimulating gingival fibroblasts, in HaCaT cells androgens promotes a shortage or inhibition of cell growth and activity. Furthermore, pharmacological treatment with ANA, FLU, FUL, and their associations to T, appears to influence cellular events of human gingival fibroblasts and HaCaT cells in vitro.
Mestre
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Al-Rikabi, Aaiad H. A. "Impaired Wound Healing and Inflammation: The Role of the Dermal Fibroblast. Phenotypic Changes in the Human Dermal Fibroblast with Inflammation; Potential Impact on Wound Healing." Thesis, University of Bradford, 2019. http://hdl.handle.net/10454/18331.
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Dermal fibroblasts positively contribute throughout the wounding response by
secreting a profile of pro- and anti-inflammatory cytokines in the wound milieu. However, a chronically inflamed environment will, cause detrimental effects on
the functional, secretory, and molecular properties of these cells. This study
aims to understand how the effect of the pro-inflammatory cytokine TNF-α
modulates both healthy and diabetic dermal fibroblast phenotype.
To mimic a chronic inflammatory environment and assess whether fibroblasts
respond similarly in different anatomical sites, donor-matched fibroblasts from
face and scalp were pre-incubated for 3 days with different concentrations (2.5,
25 or 250 ng/ml) of TNF-α. All concentrations significantly impaired proliferation
by day 14 in cells from both sites and stimulated (papillary) metabolic activity at
day 14.
However, this did not correlate with an increase in papillary cell senescence
since this did not appear until passage 17, and then only at a supra pathophysiological concentration.
Migration of dermal fibroblasts, assessed by the scratch assay. TNF-α
significantly inhibited the cells migration, particularly in diabetic fibroblasts,
suggesting they are more sensitive to TNF-α. Since TNF-α may stimulate the
secretion of soluble paracrine factors by dermal fibroblasts, conditioned medium
was collected to assess its effect on other dermal fibroblasts, however, this had
no significant effect on migration. However, using gelatin zymography, it was found that TNF-α did stimulate the
secretion of soluble paracrine factors that induce MMP activity in non-diabetic
fibroblasts, mirroring previous observations that a pro-inflammatory environment
can increase proteolytic activity, and indicating that diabetic fibroblasts were
again more sensitive than healthy. No difference was observed with MMP-9
activity and nor did the results with dermal fibroblasts reach statistical
significance, perhaps because of a relatively low n-number.
The ability of TNF-α to modulate the expression of genes associated with the
ECM (MMP-1, -2, -9, TIMP-1, and -2) and senescence (Sirt1 and 6) was
investigated. There was no change in Sirt1 and Sirt6 expression and no
evidence of paracrine effects (conditioned medium) on any of the genes. TNF-α
significantly induced mRNA expression of MMP-1 in healthy non-scratched and
scratched diabetic fibroblasts, and TIMP-1 in healthy non-scratched cells. There
was also considerable donor variability that prevented statistical significance
being achieved under the other conditions.
The secretion of various cytokines associated with inflammation was compared
in healthy and diabetic fibroblasts in the presence and absence of TNF-α.
Seven cytokines were secreted, by healthy and diabetic male and female
fibroblasts, although diabetic female fibroblasts did not secrete two of them.
TNF-α stimulated secretion of cytokines in healthy and diabetic, male and
female cells but the profiles of those released were different between the
different groups. There was no TNF-α induced paracrine effect on cytokine
secretion by healthy dermal fibroblasts.
In conclusion, changes in the microenvironment and the influx of pro inflammatory cytokines may significantly alter the dermal fibroblast phenotype.
Understanding these functional and molecular changes in response to
inflammatory cytokines will give a better understanding of the differences
between fibroblast activity in normal physiological wound healing and chronic or
diabetic non-healing wounds.
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McKnight, Holly A. "PROTEOMIC ANALYSIS OF MEMBRANE BOUND AND ASSOCIATED PROTEINS OF HUMAN GINGIVAL FIBROBLASTS AND PERIODONTAL LIGAMENT FIBROBLASTS." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338325450.
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Junker, Johan. "Human Dermal Fibroblasts in Tissue Engineering." Doctoral thesis, Linköpings universitet, Cellbiologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-19716.
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The loss or failure of tissues and/or organs is one of the most frequent problems in modern healthcare. The field of tissue engineering applies the principles of biology and engineering in order to develop functional substitutes for damaged tissues. Tissue engineering contains elements of medicine, material science and engineering with major components in focus being cells, biomaterials and soluble factors. All three components may be required for the development of clinical treatments. The usage of autologous tissue specific cells for clinical treatment is often not feasible due to poor growth kinetics or unstable phenotypes of the cells. Furthermore, lack of availability of healthy tissue that can be biopsied is a major problem in many applications. One approach to overcome this problem is to use adult stem cells which have the capacity to give rise to several different cell types. Although promising, adult stem cells have major impediments for use in several tissue engineering applications. The difficulties associated with harvest, culture and storage render problems in the development of clinically relevant procedures. During the last years, the inherent plasticity of differentiated somatic cells has been demonstrated. One of the easiest human cell types to obtain, expand and store is the dermal fibroblast. Recent reports indicate that dermal fibroblasts can be induced to differentiate towards several distinct mesenchymal lineages in vitro. The main aim of this thesis was to investigate the inherent stem cell plasticity of human dermal fibroblasts and explore their possible usefulness in tissue engineering applications. The papers included in this thesis employ routine and immunohistochemical staining, enzyme activity assay, analysis of low density lipoprotein incorporation, capillary-like network formation assay and full expression micro array analysis. Fibroblasts were shown to differentiate towards adipocyte, chondrocyte, endothelial and osteoblast-like cell types in vitro. The differentiation from fibroblasts to myofibroblasts in burn scar tissue upon stimulation by mechanical tension was also demonstrated. Adipogenic, chondrogenic and osteogenic induced fibroblasts display the upregulation of several genes associated with adipocytes, chondrocytes and osteoblasts.
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Hall, Sarah Elizabeth. "Neutrophil interactions with fibroblasts in culture." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46328.
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Karlson, Tanya De L. "Regulation of mucosal inflammation by fibroblasts /." Göteborg : Department of Microbiology and Immunology, Göteborgs universitet, 2007. http://hdl.handle.net/2077/3328.
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Vo, Annie Phuong. "Glucose Metabolism in Cancer-Associated Fibroblasts." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11025.
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Under normal conditions, non-transformed cells rely on glycolysis followed by
oxidative phosphorylation to generate ATPs. When oxygen is scarce or when cells are
actively proliferating, cellular ATPs come mainly from glycolysis. Pyruvate is converted into lactate to allow glycolysis to continue. Interestingly, cancer cells have adapted to favor lactate production even at normal oxygen tensions, exhibiting a metabolic shift known as the Warburg effect. However, the metabolic state of other cellular constituents within the tumor remains mostly unknown. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells. They aid tumor growth and metastasis by providing growth factors, cytokine, ECM remodeling proteins and interacting with other tumor stromal cells. Here I show that the Warburg effect also operates in stromal fibroblasts of the tumor microenvironment. Using mass spectrometry, genetic mouse models, gene expression and methylation studies, I demonstrate that CAFs from human and mouse mammary tumors exhibit hyperactive glycolysis and a metabolic shift towards lactate production. Furthermore, this phenotype may be sustained through epigenetic modifications of endogenous hypoxia-inducible factor 1α, key regulatory enzymes fructose-bisphosphatase 1 and pyruvate kinase M2. Depletion of stromal fibroblasts or suppression of lactate production specifically in these cells alters the metabolic profile of not only the tumors but also the cancer cells and results in impeded tumor growth. These results collectively suggest that tumor growth is dependent on metabolic state and metabolic support of stromal fibroblasts, highlighting these cells as attractive therapeutic targets in controlling cancer progression.
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Junker, Johan P. E. "Human dermal fibroblasts in tissue engineering /." Linköping : Department of Clinical and Experimental Medicine, Linköping University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-19716.
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Gagliolo, Jean-Marie. "Rôle de la sénescence des fibroblastes dans la physiopathologie de la bronchopneumopathie chronique obstructive." Thesis, Paris Est, 2013. http://www.theses.fr/2013PEST1065.
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La sénescence, perte irréversible des capacités réplicatives des cellules associée à la sécrétion de médiateurs inflammatoires, pourrait participer au développement de l'atteinte pulmonaire dans la bronchopneumopathie chronique obstructive (BPCO) en initiant, maintenant et propageant un état inflammatoire. L'objectif de ce travail était d'évaluer les mécanismes de la sénescence impliqués dans l'induction et le maintien de l'inflammation au cours de la BPCO. Ainsi, des fibroblastes pulmonaires de témoins et de patients atteints de BPCO ont été mis en culture à long terme. Un phénotype sénescent majoré associée à un sécrétome pro-inflammatoire était détectée dans les fibroblastes de patients avec BPCO par rapport aux témoins. Par ailleurs, ces fibroblastes présentaient une expression accrue des récepteurs à la PGE2 (EP2 /4)au stade non sénescent et une production accrue de PGE2, un médiateur lipidique pro-inflammatoire, au stade sénescent. Dans cette optique, une partie du travail a consisté à déterminer si la PGE2 pouvait induire la sénescence et l'inflammation des fibroblastes pulmonaires de sujets atteints ou non de BPCO. Nous avons pu démontrer que la PGE2 synthétisée par les fibroblastes sénescents induisait, maintenait (effet autocrine) et propageait (effet paracrine) la sénescence et l'inflammation associée via une voie EP2/4 / COX-2 / oxydants / p53. L'implication des oxydants dans l'induction de la sénescence nous a conduit à étudier les effets de l'hème oxygénase (HO)-1, un système anti-oxydant et anti-inflammatoire sur la prévention de la sénescence des fibroblastes pulmonaires. Ainsi, des fibroblastes pulmonaires ont été traités chroniquement avec des substances pharmacologiques modulant l'activité d'HO-1. Des résultats préliminaires nous ont permis d'observer que l'activation de HO-1 prévenait l'induction de la sénescence chez des fibroblastes pulmonaires de témoins et de BPCO. Au total, la modulation des voies de la PGE2 et de l'HO-1 pourrait contribuer à limiter la sénescence des fibroblastes pulmonaires dans la BPCOCellular senescence, a state of irreversible loss of replicative capacity associated with the secretion of inflammatory mediators, could participate in the development of chronic obstructive pulmonary disease (COPD) by initiating, maintaining and propagating an inflammatory state. The aim of this PhD project was to evaluate the mechanisms involved in senescence induction in COPD lung fibroblasts. COPD fibroblasts exhibited an increased senescent phenotype as compared to control cells. In addition, COPD fibroblasts showed an increased PGE2 receptors (EP2 /4) expression at non senescent stage and PGE2 production, apro-inflammatory lipid mediator at senescent stage. In this context, one part of the study was devoted to determine whether PGE2 could induce senescence of lung fibroblasts of subjects with and without COPD. We have shown that PGE2 synthesized by senescent fibroblasts induced, maintained (autocrine effect) and propagated (paracrine effect) senescence and associated inflammation via EP2 /4 / COX-2 / oxidants / p53 pathway. The essential role of oxidants production in the induction of senescence in COPD led us to study the effects of heme oxygenase (HO)-1, an antioxidant and anti-inflammatory system on the prevention of senescence in COPD fibroblasts. Pharmacological activation of HO-1 by hemin prevented the induction of senescence in lung fibroblasts from COPD patients probably in relation with an anti -oxidant effect. The modulation of PGE2 and HO-1 pathways may contribute to attenuate fibroblasts senescence in COPD
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Beà, Tàrrega Aida. "Caracterització de la biologia dels fibroblasts cardíacs i la seva resposta a estrès cel·lular." Doctoral thesis, Universitat de Lleida, 2021. http://hdl.handle.net/10803/671500.
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Els fibroblasts cardíacs són els principals productors de la matriu extracel·lular que cohesiona la musculatura del cor, conferint-li les propietats ideals per transformar la contracció dels cardiomiòcits en el moviment de la paret cardíaca que permet el bombeig de la sang. A més, aquestes cèl·lules intervenen en el procés de sincronització del batec i la secreció de factors tròfics potenciant el creixement del cor durant el desenvolupament. En condicions patològiques, com ara el tall del flux sanguini al miocardi (isquèmia), els fibroblasts cardíacs secreten la matriu que generarà la cicatriu durant la mort del miocardi. No obstant, tot i que aquest esdeveniment és essencial per sobreviure a l’infart, la deposició de matriu extracel·lular pot representar una complicació si s’allarga en el temps o s’activa excessivament. Per això, s’està destinant molt esforç a la identificació de l’origen embrional i els tipus cel·lulars precisos que constitueixen la població de fibroblasts cardíacs, a re-avaluar la seva abundància al cor, així com a comprendre la regulació de la síntesi i secreció de matriu extracel·lular. El nostre grup havia demostrat que els fibroblasts cardíacs expressen elevats nivells de Bcl-2, una proteïna anti-apoptòtica que els protegeix la integritat mitocondrial durant la isquèmia, reduint l’activació de les caspases. Tot i que estan ben caracteritzats pel que fa a la quantitat, l’origen genètic i l’activitat dels fibroblasts cardíacs, hi ha poca informació sobre els mecanismes implicats en la resistència d’aquestes cèl·lules a la isquèmia i per això, vam decidir seguir investigant aquest aspecte. La hipòtesi inicial era que l’autofàgia, un procés implicat en el reciclatge de components cel·lulars regulat per Bcl-2 i que pot ser utilitzat per obtenir energia, podia estar implicada en la major supervivència dels fibroblasts cardíacs a la isquèmia. L’anàlisi de l’autofàgia a fibroblasts primaris neonatals de rata, utilitzant tant inhibidors químics com el silenciament de gens clau, va mostrar que és un procés important en condicions normals però no durant la isquèmia en aquestes cèl·lules. Malgrat això, vam trobar que l’elevada expressió de Bcl-2 és necessària per la supervivència dels fibroblasts cardíacs en condicions normals, a més de durant la isquèmia i l’estrès de reticle. També vam trobar que aquestes cèl·lules presenten diverses característiques associades a la major capacitat de supervivència en relació a d’altres fibroblasts, incloent major respiració basal i de reserva, nivells de radicals lliures d’oxigen elevats però millor controlats durant la isquèmia, nivells elevats d’expressió d’enzims antioxidants i de complexos de la cadena respiratòria, però menys expressió de Pgc-1α i Mitofusina-2, associat a una xarxa mitocondrial amb més mitocondris aïllats. Aquestes característiques es troben relacionades amb la major activació de senyalització inflamatòria i vam mostrar que moltes depenen d’una activació basal elevada de la via de transducció de senyal Jak/Stat. En conjunt, els nostres resultats ajuden a la millor comprensió de les característiques biològiques subjacents a la major capacitat de supervivència dels fibroblasts cardíacs.Los fibroblastos cardíacos son los principales productores de la matriz extracelular que cohesiona la musculatura del corazón, confiriéndole las propiedades ideales para transformar la contracción de los cardiomiocitos en el movimiento de la pared cardíaca que permite el bombeo de la sangre. Además, estas células intervienen en el proceso de sincronización del latido y la secreción de factores tróficos potenciando el crecimiento del corazón durante el desarrollo. En condiciones patológicas, tales como el corte del flujo sanguíneo al miocardio (isquemia), los fibroblastos cardíacos secretan la matriz que generará la cicatriz durante la muerte del miocardio. Sin embargo, aunque este evento es esencial para sobrevivir al infarto, la deposición de matriz extracelular puede representar una complicación si se alarga en el tiempo o se activa excesivamente. Debido a eso, se está destinando mucho esfuerzo a la identificación del origen embrionario y los tipos celulares precisos que constituyen la población de fibroblastos cardíacos, a re-evaluar su abundancia en el corazón, así como a comprender la regulación de la síntesis y secreción de matriz extracelular. Nuestro grupo había demostrado que los fibroblastos cardíacos expresan niveles elevados de Bcl-2, una proteína anti-apoptótica que protege la integridad mitocondrial durante la isquemia, reduciendo la activación de las caspasas. Aunque se han caracterizado bien la cantidad, el origen genético y la actividad de los fibroblastos cardíacos, hay poca información sobre los mecanismos implicados en la resistencia a la isquemia de estas células y por eso decidimos seguir investigando este aspecto. La hipótesis inicial era que la autofagia, un proceso implicado en el reciclaje de componentes celulares regulado por Bcl-2 y que puede ser utilizado para obtener energía, podía estar implicada en la mayor supervivencia de los fibroblastos cardíacos a la isquemia. El análisis de la autofagia en fibroblastos primarios neonatales de rata, utilizando tanto inhibidores químicos como el silenciamiento de genes clave, mostró que es un proceso importante en condiciones normales, pero no durante la isquemia en estas células. Sin embargo, encontramos que la elevada expresión de Bcl-2 es necesaria para la supervivencia de los fibroblastos cardíacos en condiciones normales, además de durante la isquemia y el estrés de retículo. Encontramos también que estas células presentan varias características asociadas a la mayor capacidad de supervivencia en relación a otros fibroblastos, incluyendo mayor respiración basal y de reserva, niveles de radicales libres de oxígeno elevados pero mejor controlados durante la isquemia, niveles elevados de expresión de enzimas antioxidantes y de complejos de la cadena respiratoria, pero menos expresión de Pgc-1α y Mitofusina-2, asociado a una red mitocondrial con más mitocondrias aisladas. Estas características están relacionadas con la mayor activación de señalización inflamatoria y mostramos que muchas dependen de una activación basal elevada de la vía de transducción de señal Jak/Stat. En conjunto, nuestros resultados ayudan a una mejor comprensión de las características biológicas subyacentes a la mayor capacidad de supervivencia de los fibroblastos cardíacos.
Cardiac fibroblasts are the main producers of the extracellular matrix that binds together the cardiac muscle, giving it the ideal properties to transform the contraction of cardiomyocytes into the movement of the heart wall that allows blood pumping. In addition, these cells intervene in the process of beat synchronization and the secretion of trophic factors, enhancing the growth of the heart during development. In pathological conditions such as cut off of blood flow to the myocardium (ischemia), cardiac fibroblasts secrete the matrix that will generate the scar during myocardial death. Although this event is essential to survive the infarction, the deposition of extracellular matrix can represent a complication if it is prolonged in time or is excessively activated. That is why a lot of effort is being devoted to identifying the embryonic origin and the precise cell types that make up the population of cardiac fibroblasts, to re-evaluating their abundance in the heart, as well as to understanding the regulation of extracellular matrix synthesis and secretion. Our group had shown that cardiac fibroblasts express a high level of Bcl-2, an anti-apoptotic protein that protects mitochondrial integrity during ischemia, reducing the activation of caspases. Although the number, genetic origin, and activity of cardiac fibroblasts have been well characterized, there is little information on the mechanisms involved in the resistance of these cells to ischemia and that is why we decided to continue investigating this aspect. The initial hypothesis was that autophagy, a process involved in the recycling of cellular components regulated by Bcl-2 and that can be used to obtain energy, could be involved in the greater survival of cardiac fibroblasts to ischemia. Analysis of autophagy in neonatal rat primary fibroblasts, using both chemical inhibitors and key gene silencing, showed that it is an important process under normal conditions, but not during ischemia in these cells. However, we found that the high expression of Bcl-2 is necessary for the survival of cardiac fibroblasts under normal conditions, as well as during ischemia and reticulum stress. We also found that these cells present several characteristics associated with a higher survival capacity in relation to other fibroblasts, including greater basal and reserve respiration, elevated levels of reactive oxygen species, which are better controlled during ischemia, high levels of expression of antioxidant enzymes and of complexes of the respiratory chain, but less expression of Pgc-1α and Mitofusin-2, associated with a mitochondrial network with more isolated mitochondria. These characteristics are related to the greater activation of inflammatory signaling and we show that many of them depend on a high basal activation of the Jak/Stat signal transduction pathway. Taken together, our results help to better understand the biological characteristics underlying the greater survival capacity of cardiac fibroblasts.
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Cherutti, Giselle. "Desenvolvimento e caracterização de dispositivo de PLLA/Trietil-Citrato associado à derme suína acelular para reparação de lesões cutâneas." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/263851.
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Orientador: Eliana Aparecida de Rezende DuekDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica
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Resumo: Um dos desafios da Engenharia Tecidual é o de promover um melhor funcionamento de órgãos e tecidos danificados por doenças ou traumas. Com o objetivo de desenvolver um dispositivo bifásico para futura regeneração dérmica e aplicação na Engenharia Tecidual e Medicina Regenerativa, cultivou-se células fibroblásticas da linhagem VERO sobre uma matriz dérmica suína acelular associada a um polímero biorreabsorvível, poli (L-ácido láctico) (PLLA), com adição de um plastificante, o trietil citrato de sódio (TCS). Para a realização da pesquisa, membranas PLLA-TCS e PLLA puro foram preparadas e caracterizadas através de Microscopia Eletrônica de Varredura (MEV), Ensaio Dinâmico Mecânico pelo Módulo de Tração, Espectroscopia na região do Infravermelho com Transformada de Fourier (FTIR), Ressonância Magnética Nuclear (RMN 13C e 1H), Ângulo de Contato e Calorimetria Exploratória Diferencial (DSC). Os resultados mostraram que as membranas de PLLA- TCS, tornaram-se porosas e mais hidrofílicas em relação as membranas de PLLA puro, o que aumentou sua interação com as células fibroblásticas. Após a associação das membranas de PLLA-TCS à derme suína, as amostras foram analisadas por meio de Técnicas Histológicas e Microscopia Confocal para avaliar a presença de fibras colágenas e sua organização no arcabouço. Em seguida realizou-se cultura de células fibroblásticas sobre o dispositivo bifásico após 24 horas e 2 dias de cultivo para ensaios de Viabilidade Celular, e posteriormente Microscopia Eletrônica de Varredura (MEV). Os resultados obtidos pelos ensaios mecânicos e biológicos comprovaram que o material apresentou interação suporte-célula, principalmente devido a sua porosidade e afinidade celular apresentada pela composição estrutural da matriz dérmica, sendo atóxico às células VERO. O material atuou como substrato celular, sendo que a proliferação das células VERO foi maior em comparação com a placa de cultivo (controle), havendo infiltração celular. Concluí-se assim, que o dispositivo estudado apresenta potencial para ser utilizado como um substituto dérmico para implantes em áreas de queimaduras extensas, por ser altamente poroso, promovendo assim, maior migração, adesão e crescimento celular ao mesmo tempo em que o dispositivo é degradado pelo organismo. A capacidade de deformação apresentada pelo futuro substituto dérmico também auxilia em sua implantação, por facilitar o procedimento cirúrgico, que muitas vezes necessita distender um pouco o material para o total recobrimento da lesão; ou as movimentações naturais da pele após o implante. Além disso, o dispositivo minimiza as chances de contração do enxerto, por ser constituído por um componente dérmico
Abstract: One of the challenges of the Tissue Engineering is to promote a better functioning of organs and tissues damaged by diseases or traumas. With target for developing a biphasic device for future regeneration and dermal application in Tissue Engineering and Regenerative Medicine, fibroblasts from VERO cell line were cultivated on a porcine acellular dermal matrix associated to a bioresorbable polymer (PLLA) with the addition of a plasticizer (TCS). For this present study, PLLA-TCS membranes and pure PLLA were prepared and analyzed by means of characterization tests such as Scanning Electron Microscopy (SEM), Dynamical Mechanical Analysis (DMA), Spectroscopy of the Fourier Transform Infrared, Nuclear Magnetic Ressonance (NMR 13C e 1H), Contact Angle and Differential Scanning Calorimetry (DSC). The results showed that the PLLA-TCS, became porous and more hydrophilic compared to pure PLLA, which increased its interaction with the fibroblast cells. After the association of the PLLA-TCS to the porcine dermal matrix, the samples were analyzed by Histological Techniques and Confocal Microscopy to evaluate the presence of collagen fibers and their organization within scaffold. Afterwards, a culture of fibroblasts cell on the biphasic device was performed after 2 days and 24 hours of cultivation the Cellular Viability test was done and posteriorly Scanning Electron Microscopy (SEM). The results of the biphasic device in relation to mechanical and biological tests showed the cell-support interaction, through the analysis of viability, cell morphology and structural organization of collagen fibers and polymer structure, are nontoxic to VERO cells. The material behaves as a cell substrate where proliferation of VERO cells and their infiltration was higher compared to the cell culture plate. It can therefore be concluded that the studied device has the potential to be used as a substitute for dermal implants in areas of extensive burns, for being highly porous, thus promoting increased migration, adhesion and cell growth while the device is degraded by the body. The device deformation capacity also helps in the substitute implantation for facilitating the surgical procedure which often need to stretch the material for coverage of the injury completely or natural movements of the skin after implantation. Furthermore, the device minimizes the chances of skin graft contraction as the gadget consists of a dermal component
Mestrado
Materiais e Processos de Fabricação
Mestre em Engenharia Mecânica
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Silva, Patrícia Lacouth da. "Mecanismos celulares e teciduais da regeneração em holotúrias (Echinodermata:Holothuroidea)." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/41/41135/tde-20012012-090245/.
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Equinodermos são bem conhecidos pelas suas grandes capacidades regenerativas, principalmente em processos mais complexos como a reposição de membros e órgãos. Porém pouco se sabe sobre processos aparentemente mais simples como a cicatrização. Tem sido descrito que um dos principais mecanismos envolvidos neste evento é o remodelamento da matriz extracelular e que existem células responsáveis por isto. Entretanto, vários detalhes do processo e do exato papel destas células ainda não são muito claros. Neste trabalho, foram estudados os estágios do processo de cicatrização em Holothuria grisea Selenka, 1867 (Holothuriidae). A lesão foi induzida através de uma incisão perfurando a parede do corpo do animal até alcançar a cavidade celomática. Através de análises histológicas, foram acompanhadas as mudanças estruturais e os mecanismos celulares que ocorrem no intervalo de doze horas a trinta dias após a injúria. Foi observado que houve um rápido fechamento da ferida através da síntese de novas fibras de colágeno, e que fibroblastos e duas populações de esferulócitos estão envolvidos neste processo. São discutidas as prováveis funções destes tipos celulares e possíveis diferenças funcionais entre os esferulócitos.Echinoderms are well known by their regenerative capabilities, mostly in complex events such as the reconstruction of internal organs or entire body parts. However, the knowledge on apparently simpler processes such as wound healing is relatively scarce. It has been described that extracellular matrix remodeling is the main mechanism, and it involves participation of different cells populations. However, the specific function of these cell types is still under debate. In this work, the wound healing process in Holothuria grisea Selenka, 1867 (Holothuriidae) was studied. The lesion was made by an incision through the body wall up to the coelomic cavity. The local changes in cell types and the reorganization of the extracellular matrix were accompanied from 12 hours to 30 days after the injury. Soon after the wound, the organism responded with a localized contraction, followed by cell migration and synthesis of new collagen fibers. Four different cell types were observed, including two different types of spherulocytes. The probable functions of these cells are discussed.
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Paulino, Carmen Emilia Caba. "Efeito do protocolo de desmineralização por ácido cítrico na área de superfície radicular recoberta por fibroblastos do ligamento periodontal humano: estudo à microscopia eletrônica de varredura." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/25/25146/tde-13102014-152339/.
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A biomodificação radicular empregando ácido cítrico tem sido utilizada visando à reinserção dos tecidos periodontais a raízes expostas à doença periodontal. Entretanto, a grande diversidade metodológica entre os estudos ainda não possibilitou o estabelecimento de um protocolo amplamente aceito quanto à concentração e tempo de aplicação do ácido. Assim, 32 dentes extraídos por doença periodontal avançada forneceram 63 fragmentos radiculares que, após raspagem manual, foram divididos nos seguintes grupos de tratamento: Grupo AC-10-90: desmineralização com ácido cítrico a 10% em pH 1, durante 90 segundos; Grupo AC-10-120: desmineralização com ácido cítrico a 10% em pH 1, durante 120 segundos; Grupo AC-10-180: desmineralização com ácido cítrico a 10% em pH 1, durante 180 segundos; Grupo AC-50-90: desmineralização com ácido cítrico a 50% em pH 1, durante 90 segundos; Grupo AC-50-120: desmineralização com ácido cítrico a 50% em pH 1, durante 120 segundos; Grupo AC-50-180: desmineralização com ácido cítrico a 50% em pH 1, durante 180 segundos; Grupo C (controle): lavagem com soro fisiológico. Sobre as superfícies tratadas foram cultivados fibroblastos do ligamento periodontal humano por 24, 48 e 72 horas. A ampliação dos túbulos dentinários, morfologia celular e a porcentagem das superfícies radiculares recobertas por células foram avaliadas em microscopia eletrônica de varredura. As imagens microscópicas das superfícies recobertas por células foram comparadas pelo teste não paramétrico de Kruskal-Wallis seguido pelo teste de Dunn e na ampliação dos túbulos pelo teste de variância a dois critérios (ANOVA) complementado pelo teste de Tukey, em 5% de significância, ambos realizados por um programa computadorizado comparando os resultados entre os grupos. A Com exceção do grupo C, em todos os grupos houve aumento crescente da cobertura da superfície radicular por fibroblastos com o tempo. A maior área de cobertura foi apresentada pelo grupo AC-10-90 (98,82±2,57%) às 24 horas e essa diferença foi significante (p<0,001) em comparação aos grupos AC-50-90 (64,94±20,60%), AC-50-180 (56,59±35,42%) e C (0,06±0,24%). Nas demais comparações de tempo de aplicação e tempo de cultura, predominou a superioridade dos grupos tratados por ácido cítrico a 10% sobre os de 50%, porém, sem significância estatística. Todos os grupos teste foram significantemente superiores aos controle em todos os tempos de cultura. O menor valor médio para o diâmetro dos túbulos dentinários expostos pelos tratamentos foi apresentado pelo grupo AC-10-90 (4,55±0,69 μm) que diferiu significantemente (p<0,001) dos grupos AC-10-120 (5,33±0,95 μm), AC-10-180 (5,54±1,56 μm) e AC-50-180 (5,56±1,22 μm). Esse último apresentou a maior ampliação, porém sem diferença significante em relação aos demais grupos. Os fibroblastos apresentaram-se mais espalhados, achatados e com menor definição de limites nos grupos tratados com ácido cítrico a 10% do que nos de 50%, cujas células apresentavamse fusiformes e arredondadas. Concluiu-se que o ácido cítrico a 10% por 90 segundos produziu superfície mais favorável à proliferação celular com características morfológicas de estágios mais avançados de diferenciação e área de cobertura superficial por fibroblastos mais extensa no período inicial de cultura do que na concentração de 50%. A ampliação dos túbulos dentinários pareceu não influenciar a cobertura superficial por fibroblastos. Estudos subsequentes devem investigar a influência das propriedades químicas do agente biomodificador radicular para contribuir para a elucidação das diferenças produzidas no comportamento celular.The root biomodification employing citric acid has been used in order to reattach periodontal tissues to root surface exposed to periodontal disease. However, the methodological diversity among studies has not allowed the establishment of a widely accepted protocol as the concentration and time of acid application. Thus, 32 teeth were extracted due to advanced periodontal disease, so that 63 root fragments were provided. After scaling and root planning, the fragments were divided into the following treatment groups : AC-10-90 group: demineralization with 10% citric acid at pH 1 for 90 seconds; AC-10-120 group: demineralization with 10% citric acid at pH 1 for 120 seconds; AC-10-180 group: demineralization with 10% citric acid at pH 1 for 180 seconds; AC-50-90 group: demineralization with 50% citric acid at pH 1 for 90 seconds; AC-50-120 group: demineralization with 50% citric acid at pH 1 for 120 seconds; AC-50-180 group: demineralization with 50% citric acid at pH 1 for 180 seconds; group C (control) : rinsing with saline solution. On the treated surfaces, fibroblasts from human periodontal ligament were cultured by 24, 48 and 72 hours. The enlargement of the dentinal tubules, cell morphology and the percentage of root surface covered by cells were evaluated by scanning electron microscopy. Those microscopic images from the root surfaces covered by cells were compared by the nonparametric Kruskal-Wallis test followed by Dunn\'s test and the enlargement of tubules by two variance (ANOVA) complemented by the Tukey test, both performed by a computer program comparing the results between the groups, at 5% significance. With the exception of group C, all groups showed increasing coverage of the root surface by fibroblasts over time. The largest area of coverage was presented by AC-10-90 (98.82±2.57%) at 24 hours and this difference was significant (p <0.001) compared to AC-50-90 (64.94±20.60%), AC-50-180 (56.59±35.42%) and C (0.06±0.24%). In other comparisons the application time and culture time groups treated with citric acid at 10% were superior to groups of 50%, without statistical significance. All test groups were significantly better than the control ones at all times of culture. The shortest average diameter of dentinal tubules exposed by the treatments was presented by AC-10-90 (4.55±0.69 μm) group that differed significantly (p<0,001) from AC-10-120 (5 groups 33±0.95 μm), AC-10-180 (5.54±1.56 μm) and AC-50-180 (5.56±1.22 μm). This last showed the highest enlargement, but without significant difference compared to the other groups. The fibroblasts were more spread, flattened and had less identifiable limits in the groups treated with citric acid 10% than those in 50% which cells had become rounded and spindle. It was concluded that the demineralization with 10% citric acid for 90 seconds produced more favorable surface to cell proliferation, more morphological characteristics of later stages of differentiation and larger surface area coverage by fibroblasts in the initial periods of culture than any of the groups treated with 50% citric acid. The enlargement of dentinal tubules did not seem to influence the surface coverage by fibroblasts. Subsequent studies should investigate the influence of the chemical properties of the root conditioner agent on the root surfaces in order to contribute to the elucidation of the differences produced in the cell behavior.
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Maker, Garth Lucas. "Regulation of surfactant production by fetal type II pneumocytes and characterization of fibroblast-pneumocyte factor /." Access via Murdoch University Digital Theses Project, 2007. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20080430.141113.
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Terron, Canedo Nuria. "miRNAs in equine sarcoids : identification and profiling of miRNAs in equine fibroblasts and BPV-1 transformed equine fibroblasts." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8130/.
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Popovic, Ana. "Étude de la conversion des fibroblastes en fibroblastes associés au cancer au sein du microenvironnement tumoral des mélanomes cutanés." Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://theses.univ-cotedazur.fr/2021COAZ6011.
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Aux stades avancés, le mélanome est le cancer de la peau le plus agressif en raison de son fort potentiel métastatique et sa résistance aux traitements. Malgré les progrès récents réalisés dans sa prise en charge, environ 50 % des patients atteints de mélanome avancé sont en impasse thérapeutique. Une stratégie prometteuse consiste à cibler les fibroblastes associés au mélanome (MAFs) présents dans le microenvironnement tumoral (TME), car les fibroblastes activés par le mélanome sont reconnus comme des acteurs clés dans la tumorigenèse, les métastases et la résistance thérapeutique. La compréhension du ou des mécanismes moléculaires responsables de la transition phénotypique des fibroblastes en MAFs permettra d’élaborer de nouvelles stratégies afin de les inhiber et améliorer le taux de guérison des stades avancés. La majorité des MAFs présentent des propriétés myofibroblastiques et expriment la protéine d'activation des fibroblastes α (FAPα). Cependant, le rôle de FAPα dans la transition et le maintien du phénotype MAF ainsi que les processus tumoraux contrôlant son expression sont très peu connus. Mon travail repose sur l’étude de la régulation et du rôle de FAPα dans le TME du mélanome. Dans ce contexte, nous avons investigué les molécules de signalisation qui règlent le passage phénotypique des fibroblastes cutanés en MAFs et le rôle de FAPα dans le phénotype MAF et la progression du mélanome. Nos résultats montrent que le milieu conditionné (CM) provenant d'un mélanome invasif conduit les fibroblastes à adopter un phénotype tumoral avec une augmentation des marqueurs moléculaires de MAFs comme αSMA, intégrine β1 et FAPα et pro-invasifs comme YAP. De plus, les fibroblastes traités avec le CM de mélanome invasif présentent des caractéristiques myofibroblastiques, comme la capacité à produire une matrice extracellulaire (ECM) de fibres de collagène et de fibronectine alignées. L'inhibition pharmacologique de la voie de signalisation du TGFβ1 montre que le TGFβ1 est le principal facteur sécrété par les cellules invasives, qui induit la transition phénotypique en MAF. Fait important, les propriétés myofibroblastiques des MAFs dépendent de l'expression de FAPα. L’inhibition de FAPα dans les MAFs, diminue l'expression des marqueurs moléculaires proinvasifs et supprime leur capacité à produire une matrice fibrillaire alignée. Nous avons par la suite établi un modèle cellulaire dans lequel FAPα est surexprimée dans les fibroblastes normaux. La surexpression de FAPα favorise l'expression de marqueurs de MAFs, les propriétés contractiles et le dépôt de fibres d’ECM alignées. De plus, la culture de cellules de mélanome sur des matrices 3D dérivées de fibroblastes sur-exprimant FAPα diminue leur prolifération et augmente leur migration. Des observations inverses sont obtenues lorsque les cellules de mélanome sont cultivées sur des matrices dérivées de MAFs éteints pour FAPα. En conclusion, nous montrons que TGFβ1 sécrété par des cellules de mélanome invasives est responsable de l'activation des fibroblastes cutanés en un phénotype MAF exprimant FAPα, qui s'accompagne d'une activité myofibroblastique accrue qui est importante pour le remodelage matriciel du stroma tumoral. En outre, ce remodelage de l'ECM contribue au comportement invasif et agressif du mélanome. Les travaux futurs porteront sur les cibles en aval de FAPα afin de faire la lumière sur son rôle dans le TME des mélanomesAdvanced melanoma is the most aggressive skin cancer and is notorious for its tendency to metastasise. Approximately 50% of patients with advanced melanoma experience relapse due to its intrinsic resistance to current therapies. A promising therapeutic strategy is targeting melanoma associated fibroblasts (MAFs) in the melanoma tumour microenvironment (TME) because MAFs, have been implicated as key players in melanoma progression, metastasis and drug resistance. An understanding of the molecular mechanism(s) responsible for the phenotypic switching of skin fibroblasts to MAFs will enable more diverse ways of inhibiting them. In this regard, we are interested in observations that MAFs exhibit myofibroblast properties and that they express fibroblast activation protein α (FAPα). However, whether FAPα is a key player in driving the MAF phenotype and what regulates its expression are poorly understood. To address this, we investigated: 1) the signalling molecules that regulate the phenotypic switching of skin fibroblasts to MAFs and 2) the role of FAPα in the MAF phenotype and melanoma progression. Our results show that conditioned medium (CM) from invasive melanoma stimulated skin fibroblasts to adopt a MAF phenotype, as we observed an increase in mRNA and protein levels of MAF molecular markers αSMA, β1integrin and FAPα. Moreover, skin fibroblasts treated with invasive melanoma CM exhibited myofibroblast characteristics, including the deposition of aligned ECM fibres (fibronectin and collagen) and an increase in protein expression of proinvasive molecular markers such as fibronectin, collagen I and YAP. Importantly, chemical inhibition of transforming growth factor β1 (TGFβ1) in the melanoma CM demonstrated that TGFβ1 is the key signalling molecule secreted by invasive melanoma cells, which is inducing the phenotypic switching of skin fibroblasts to MAFs. Importantly, we demonstrated that MAF myofibroblast properties are dependent on FAPα expression. Our data suggest that silencing FAPα in MAFs, significantly decreased CAF molecular and proinvasive marker expression and abolished their ability to deposit aligned ECM fibres. To further investigate the role of FAPα in the MAF phenotype, we established a cell culture model in which FAPα was overexpressed in skin fibroblasts. FAPα overexpression in skin fibroblasts promoted protein expression of several MAF and proinvasive markers, contractile capacity and the deposition of aligned ECM fibres. Interestingly, we observed a decrease in melanoma cell proliferation and an increase in their migration on aligned 3D matrices derived from FAPα overexpressing skin fibroblasts and the opposite occurred when melanoma cells were cultured on matrices derived from FAPα-silenced MAFs. We therefore concluded that TGFβ1 secreted by invasive melanoma cells is responsible for activating skin fibroblasts to a FAPα-expressing MAF phenotype, which is accompanied by enhanced myofibroblast activity and is important for ECM remodelling in the TME. Furthermore, this ECM remodelling is contributing to the aggressive melanoma behaviour. Future work will investigate the downstream targets of FAPα to shed light on its role in the melanoma TME
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Wei, Shan. "Mechanisms of cellular senescence in human fibroblasts /." View online version; access limited to Brown University users, 2005. http://wwwlib.umi.com/dissertations/fullcit/3174691.
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Derhami, Kalal. "Interactions of human skin fibroblasts with titanium." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape3/PQDD_0011/NQ59577.pdf.
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Pauw, Monique Thérèse Maria van der. "Fibroblasts and their role in periodontal regeneration." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2000. http://dare.uva.nl/document/55696.
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Boright, Andrew Pepler. "Prolidase deficiency : studies in human dermal fibroblasts." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75956.
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Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.
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Hosein, Abdel Nasser. "Fundamental investigations into breast carcinoma-associated fibroblasts." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107576.
Full textAbstract:
Breast cancer stroma is a heterogenous mixture of cells types which, in addition to the carcinomatous epithelium itself, consists predominately of endothelium, adipocytes, immune infiltrates and fibroblasts. Recently there has been a movement towards characterizing each of these cells types found within the cancer stroma and understanding how they ultimately impact the disease. The cancer-associated fibroblast (CAF) has garnered a great deal of this attention and numerous in vivo models models have shown the predominance of several pathways CAFs may regulate in order to promote breast cancer progression. Despite the excitement generated by these early investigations into CAFs there has never been an attempt to comprehensively characterize human breast CAFs. It is therefore the hypothesis of thisthesis that the rational design of anti-CAF therapeutics is contingent upon the development of a molecular taxonomy of human breast CAFs. The first objective of this thesis was to aid in the resolution of a longstanding debate within the CAF community pertaining to the nature of DNA-level changes in CAFs. We found that such changes in CAFs are very rare, occurring in only 4% of patients. Interestingly, the very rare changes included a TP53 mutation which conceivably led to widespread chromosomal instability. The second objective was to use microarray technology in order to establishthe above mentioned CAF molecular taxonomy. In our panel of primary human breast CAFs we found that CAFs segregated into one of two groups. The first group tightly clustered with bone marrow-derived mesenchymal stem cells and was composed of CAFs that were almost entirely derived from breast cancers of high histological grade. The second group of CAFs clustered very closely with normal breast fibroblasts and were more often derived from low grade breast cancers. We developed a 186 gene signature that distinguished these two subtypes and showed biological significance inindependent whole breast tumor microarray datasets. Interestingly, the group of patients corresponding to the latter group of CAFs showed a poorer overall survival which we postulated may be due to an epithelial to mesenchymal transition in more aggressive breast carcinomas. The final objective was to develop a functional in vitro co-culture model between a breast cancer cell line and CAFs based on information from the expression profile study. To that end, a group of CAFs were deemed to be positive for a type one interferon (IFN) response and were capable of imparting a pro-cancer effect on the MCF-7 breast cancer cell line in a co-culture model. This effect was largely abolished through assays designed to neutralize the IFN ligand. In addition, the interferon response was correlated to a poorer survival in an independent dataset. We were also able to deduce a putative tumor suppressor gene which could be used as marker of response to future anti-IFN based therapy. Together, these findings have begun to elucidate the inter-patient heterogeneity that exists between CAFs which will greatly aid in the development of future anti-stromal based cancer therapies.Le stroma de cancer du sein est un mélange hétérogène de types de cellules qui, en plus de l'épithélium carcinomateuse lui-même, se compose essentiellement de l'endothélium, les adipocytes, infiltrats immunitaire et les fibroblastes. Récemment il ya eu un mouvement vers la caractérisation de chacun de ces types de cellules présentes dans le stroma du cancer et de comprendre leur impact sur la maladie. Les fibroblastes associé au cancer (FAC) one reçu beaucoup de cette attention et plusieurs modèles in vivo ont montré la prédominance des FACs en la promotion du cancer du sein. Malgré l'enthousiasme suscité par ces études sur des FACs, il n'a jamais été un effort d'etablir une taxonomie des FACs du sein. Il est donc l'hypothèse de cette thèse que la découverte rationnelle des thérapeutiques contre des FACs est dépendante sur une taxonomie moléculaire des FACs du sein. Le premier objectif de cette thèse était d'aider à la résolution d'un débat dans la communauté CAF concernant des changements d'ADN dans les FACs. Nous avons constaté que de tels changements dans les FACs sont très rares, se produisent dans seulement 4% des patients. Aussi, les changements très rare comprenait une mutation TP53, qui pouvait contribué à l'instabilité chromosomique. Le deuxième objectif était d'utiliser la technologie des biopuces pour d'établir le taxonomie moléculaires des FACs. Dans notre groupe de FACs primaires du sein nous avons observé que les FACs était séparés en deux groups par les expériences avec des biopuces. Le premier groupe était groupé avec des cellules dérivées de la moelle osseuse et était composé de FACs qui étaient presque entièrement dérivées de cancers du sein de haut grade histologique. Le deuxième groupe des FACs avait un profil d'expression très similaire aux fibroblastes mammaires normales et ils étaient dérivées de cancers du sein de bas grade histologique. De plus, nous avons développé une signature de 186 gènes qui distingue ces deux sous-types des FACs et a montré l'importance biologique dans des ensembles biopuce indépendantes de cancer du sein. L'objectif final était de utiliser I'information du biopuces pour développer une modèle in vitro entre une lignée cellulaire de cancer du sein et des FACs. À cette fin, un groupe de FAC ont été jugées positives pour la réponse, de type un, d'interféron (IFN). Cette réponse était capables de conférer un effet 'pro-cancer' sur la lignée cellulaire du cancer du sein MCF-7 dans un modèle de co-culture. Cet effet a été largement aboli grâce à des tests visant à eutraliser le ligand IFN. En plus, la réponse interféron était corrélée à un faible taux de survie dans un ensemble indépendants de biopuce. Nous avons trouvé un gène suppresseur de tumeur putatif qui pourrait être utilisé comme marqueur de la réponse de la thérapie contre l'interféron. Ensemble, ces résultats ont commencé à élucider l'hétérogénéité qui existe entre les FACs des patients de cancer du sein. Éventuellement cela aidera à exploiter le stroma pour l'élaboration des thérapies contre le cancer du sein.
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Elyaderani, Parisa Javadian. "Reprogramming of fibroblasts by the Piwil2 gene." Thesis, University of Newcastle Upon Tyne, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613436.
Full textAbstract:
The Piwil2 gene belongs to the Piwi family of genes conserved during evolution from Arabidopsis to human. The Piwi family genes are considered as stem cell modulators functioning in meristem cell division in Arabidopsis and germ stem cell propagation in C.elegans and in mammals. Although some essential functions such as germ cell development, transposition repression, epigenetic modification and translational regulation, as well as stem cell and cancer stem cell maintenance, are attributed to this family of genes, the detailed mechanism of the function of these genes still remains elusive. In this study, by taking advantage of the gam of function technique, the coding sequence of the Piwil2 gene was introduced into fibroblasts and stable Piwil2 expressing fibroblasts were established. These cells were evaluated for expression of germ cell specific markers, since the Piwil2 gene is well known as a regulator of spermatogenesis. Piwil2 transfected cells did not express specific markers of spermatogenesis, except Stra8 and Fragilis, but they did express the pluripotent markers of Oct4, Nanog, c-Myc and Klf4 instead. Furthermore, Piwil2 transfected cells exhibited a high tendency to form colony-like structures, partial staining for alkaline phosphatise (AP) activity, expression of germ layer markers and markers of the blood lineage. In terms of teratoma formation, Piwil2 transfected cells showed tumour outgrowth when injected subcutaneously in SCID mice. However, histological observations of the tumour sections revealed that tumours were not teratomas; instead, they were highly malignant tumours with rare signs of differentiation. This result is consistent with the suggestion that the Piwil2 gene is a cancer stem cell gene that is ectopically expressed in a variety of mammalian tumours. Although these results do not show full germ cell or i~S cell conversion of fibroblasts, the dramatic changes that were induced in the Piwil2 transfected cells, compared to the control cells, suggest this gene can act as a modulator of fundamental pathways such as proliferation and differentiation and bestow an unusual phenotype on fibroblasts. It is worth pointing out that some of the findings of this study, such as Oct4 and Terl19 expression in transfected fibroblasts, were not stable in spite of stable expression of the Piwil2 gene. The underlying reason for this might be some sort of dynamic effect of the Piwil2 gene on cell behaviour
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Lim, Kue Peng. "Fibroblasts in human oral squamous cell carcinoma." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503859.
Full textAbstract:
The tumour microenvironment is known to play an important role in tumour development and progression. The diversity and role of stromal fibroblasts in human oral cancer, however, is unknown. In this study, fibroblasts were oral cancer, however, is unknown. In this study, fibroblasts were isolated from cultures of normal oral mucosa, oral epithelial dysplasia and mortal and immortal oral carcinomas, the latter malignancy being genetically unstable. Using global gene expression profiling, we demonstrated that fibroblasts clustered according to their tissue of origin.
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Mitchell, Stephen Andrew. "The radiation response of human dermal fibroblasts." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392193.
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Cosulich, Sabina Chiara. "Modulators of the cell cycle in fibroblasts." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259439.
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Cassady, John P. "Transdifferentiation of fibroblasts to neural stem cells." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/83634.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The developmental process is carefully controlled by transcriptional and epigenetic changes that occur as a zygote transforms into an adult organism. This process can be reversed by the overexpression of transcription factors Oct4, Sox2, Klf4, and c-Myc, which reprogram a differentiated cell!s nucleus to one that is transcriptionally and epigenetically indistinguishable from an embryonic stem (ES) cell. However, it is still unclear if transcription factors can completely convert the nucleus of a differentiated cell into that of a distantly related somatic cell type with complete transcriptional and epigenetic reprogramming maintained in the absence of exogenous factor expression. To test this idea, we generated doxycyline (dox)-inducible vectors encoding neural stem cell-expressed factors. We found that stable, self-maintaining NSC-like cells could be induced under defined growth conditions. These cells were characterized in the absence of exogenous factor induction and were shown to be transcriptionally, epigenetically, and functionally similar to endogenous embryonic cortical NSCs. Additionally, a cellular system was created for reproducible generation of doxindependent iNSCs without additional factor transduction. Our results show that a transcriptionally and epigenetically reprogrammed somatic nucleus can be stabilized in vitro and provides a tool to study the mechanism of somatic cell conversion.
by John P. Cassady.
Ph.D.