Tesis sobre el tema "Colon fibroblasts"

京都大学
0048
新制・課程博士
博士(医学)
甲第8550号
医博第2272号
新制||医||747(附属図書館)
UT51-2000-M14
京都大学大学院医学研究科内科系専攻
(主査)教授 鍋島 陽一, 教授 月田 承一郎, 教授 千葉 勉
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DAM

Situés dans les cordons de Billroth de la rate, les macrophages de la pulpe rouge (RPM) sont continuellement exposés au flux du sang. Leur fonction principale est d’éliminer les globules rouges sénescents et de recycler le fer contenu dans leur hémoglobine. Les cellules dites« stromales » sont des cellules de soutien qui constituent des niches pour les macrophages de différents organes. Au cours de mon travail de thèse, j’ai étudié les mécanismes qui régulent l'homéostasie des RPM en me focalisant sur la contribution des cellules stromales. En utilisant différentes techniques d’imagerie, j’ai révélé que les RPM sont enchâssés dans un réseau réticulaire de fibroblastes caractérisés par l'expression de la protéine de la tumeur de Wilm 1(WT1) et le facteur de stimulation des colonies de macrophages 1 (CSF1). La délétion conditionnelle du gène Csf1 dans les fibroblastes de la pulpe rouge exprimant WT1, mais pasdans les fibroblastes de la pulpe blanche, réduit drastiquement le nombre de RPM sans modifier le niveau de CSF1 circulant. Suite à la déplétion des RPM, les fibroblastes de la pulpe rougeproduisent de manière transitoire les facteurs chimiotactiques pour les monocytes CCL2 et CCL7et de fait contribuent à la reconstitution de la population de RPM. Ainsi, les fibroblastes de la pulpe rouge soutiennent et nourrissent les RPM, une fonction qui est vraisemblablement conservée chez l'homme
Located within red pulp cords, splenic red pulp macrophages (RPM) are constantly exposed tothe blood flow, clearing senescent red blood cells (RBC) and recycling iron from hemoglobin.Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement ofstromal cells as these cells perform anchoring and nurturing macrophage niche functions inlymph nodes and liver. Microscopy revealed that RPM are embedded in a reticular meshwork ofred pulp fibroblasts characterized by the expression of Wilm’s Tumor 1 (WT1) and colonystimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but notwhite pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocytechemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPMnetwork. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved inhumans

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Este trabalho investigou o processo de reparação de uma ferida cirúrgica do cólon distal de ratos tratados com etanol. Foi utilizado o estudo da força de ruptura, avaliação histológica, quantificação de fibroblastos e análise morfométrica da área da cicatriz. Foram utilizados 126 ratos distribuídos por sorteio em 02 grupos: Grupo A1-controle e Grupo A2- etanol. Os animais do grupo A1 receberam ração e água “ad libitum” por 60 dias, após este período 12 animais foram sacrificados e submetidos ao estudo da força de ruptura. Os demais foram operados e submetidos a uma anastomose do cólon distal e estudados no 7o, 14o e 21o dias pósoperatório. Os animais do grupo A2 receberam ração e solução hidroalcoólica a 30% por 60 dias. Após este período foram submetidos ao mesmo procedimento do grupo controle; receberam ração e água no período pósoperatório, até o final do experimento. Verificou-se que a força de ruptura do segmento intestinal estudado foi menor em animais não operados tratados com etanol (A2M0) e, também foi menor nos animais tratados com etanol, operados e estudados no 14o dia pós-operatório (A2M2) quando comparados ao controle. A análise morfométrica da área da cicatriz demonstrou valores significantemente maiores nos animais tratados com etanol e estudados no 7o dia pós-operatório (A2M1) quando comparados ao controle...
This study investigated the healing process of a distal colon surgical wound in rats treated with ethanol. The rupture strength, histological evaluation, fibroblast quantification and morphometric analysis of the healing tissue were performed. One hundred and twenty-six rats were randomized into 2 groups: Group A1 – control group and Group A2 – ethanol group. Group A1 animals received food and water ad libidum for 60 days, after this period, 12 animals were sacrificed and underwent an evaluation of rupture strength. The remaining rats were operated and underwent distal colon anastomosis and were evaluated on the 7th /14th and 21st postoperative days. Group A2 animals received food and hydroalcoholic solution at 30% for 60 days. After this period they underwent the same procedure used for the control group. In the postoperative period they received food and water until the end of the investigation. It was observed that the rupture strength of the studied intestinal segment was lower in the animals which were not operated and treated with ethanol (A2T0). This strength was also lower in the animals which were operated and treated with ethanol evaluated on the 14th postoperative day (A2T2), when compared to the control group... (Complete abstract, click electronic access below)

Orientador: Shoiti Kobayasi
Resumo: Este trabalho investigou o processo de reparação de uma ferida cirúrgica do cólon distal de ratos tratados com etanol. Foi utilizado o estudo da força de ruptura, avaliação histológica, quantificação de fibroblastos e análise morfométrica da área da cicatriz. Foram utilizados 126 ratos distribuídos por sorteio em 02 grupos: Grupo A1-controle e Grupo A2- etanol. Os animais do grupo A1 receberam ração e água "ad libitum" por 60 dias, após este período 12 animais foram sacrificados e submetidos ao estudo da força de ruptura. Os demais foram operados e submetidos a uma anastomose do cólon distal e estudados no 7o, 14o e 21o dias pósoperatório. Os animais do grupo A2 receberam ração e solução hidroalcoólica a 30% por 60 dias. Após este período foram submetidos ao mesmo procedimento do grupo controle; receberam ração e água no período pósoperatório, até o final do experimento. Verificou-se que a força de ruptura do segmento intestinal estudado foi menor em animais não operados tratados com etanol (A2M0) e, também foi menor nos animais tratados com etanol, operados e estudados no 14o dia pós-operatório (A2M2) quando comparados ao controle. A análise morfométrica da área da cicatriz demonstrou valores significantemente maiores nos animais tratados com etanol e estudados no 7o dia pós-operatório (A2M1) quando comparados ao controle... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This study investigated the healing process of a distal colon surgical wound in rats treated with ethanol. The rupture strength, histological evaluation, fibroblast quantification and morphometric analysis of the healing tissue were performed. One hundred and twenty-six rats were randomized into 2 groups: Group A1 - control group and Group A2 - ethanol group. Group A1 animals received food and water ad libidum for 60 days, after this period, 12 animals were sacrificed and underwent an evaluation of rupture strength. The remaining rats were operated and underwent distal colon anastomosis and were evaluated on the 7th /14th and 21st postoperative days. Group A2 animals received food and hydroalcoholic solution at 30% for 60 days. After this period they underwent the same procedure used for the control group. In the postoperative period they received food and water until the end of the investigation. It was observed that the rupture strength of the studied intestinal segment was lower in the animals which were not operated and treated with ethanol (A2T0). This strength was also lower in the animals which were operated and treated with ethanol evaluated on the 14th postoperative day (A2T2), when compared to the control group... (Complete abstract, click electronic access below)
Mestre

Colorectal cancer (CRC) is the third most common cancer in Western Countries and one of the leading causes of mortality (WHO). CRC is a multistep process involving hyperproliferation of normal epithelial cells, due to an Apc gene mutation, and proceeding through adenomas and adenocarcinomas development due to progressive accumulation of mutations on oncogenes and oncosuppressor genes. These mutations can be due, among others, to increased genomic instability, oxidative stress, and epigenetic changes. Among this last one, DNA methylation changes in CpG islands are involved in 18% of CRC cases (Gallois et al. 2016). Moreover, progress and invasion of CRC are also stimulated by the microenvironment: Cancer Associated Fibroblasts (CAFs) are its main component, responsible for the epithelialmesenchymal transition and inflammation promotion (Wang et al. 2017). A wide range of data are present in literature about CAFs role in colon carcinogenesis: nevertheless, the link between Apc mutation and colon tissue microenvironment has not been clearly defined yet. Three aims have characterized this PhD project: first, the study of natural chemopreventive strategies capable of interrupting CRC carcinogenesis with associated very low toxicity. Indeed, the use of NSAIDs, showed to be a promising chemopreventive strategy in the past decades, is limited by their possible side effects: instead, the use of natural products could allow to overtake this limitation and be useful for secondary and tertiary prevention in individuals at high risk of CRC development. Three different natural compounds/products (a bergamot juice extract from endocarp (BJe), morin, and a pomegranate mesocarp decoction (PMD)) were tested in vivo in the Pirc rat model (F344/NTac-Apcam1137), bearing an Apc gene mutation and spontaneously developing colorectal polyps (Amos-Landgraf et al. 2007) and microscopic preneoplastic lesions (MDFs, Femia et al. 2015) in the normal mucosa (NM): MDFs are also considered useful endpoint for short-term chemopreventive studies. In addition, these in vivo experiments were supported by in vitro tests, aimed to explain the molecular mechanisms of the 3 tested compounds. The second aim was the study of the link between Apc gene mutation and tissue microenvironment at very early stages of colon carcinogenesis, addressing the role of colon fibroblasts within tissue microenvironment, at a stage in which colon carcinogenesis is already ongoing but no macroscopic lesions can be found. We evaluated the effects of this mutation on colon fibroblasts phenotype in established primary cultures from the colon of Pirc and F344-Wt age-matched (one month) rats. The third aim was to add knowledge on the role of Apc mutation on DNA stability at early stages of colon carcinogenesis, with the use of the COMET assay to evaluate DNA strand breaks and oxidative damage in both cultured colon fibroblasts of Pirc and Wt rats and in apparently morphological normal mucosa samples of Pirc and Wt rats aged one month. In addition, we set up a modification of the method, aiming at evaluating the global DNA methylation status. The effects of the treatments tested in Pirc rats were evaluated assessing the number of MDFs, apoptosis and proliferation both histologically and measuring relevant genes and/or proteins expression. Established Pirc and Wt colon fibroblasts primary cultures were characterized for their proliferative and phenotypic profile: both inflammatory and senescence-associated markers were evaluated with immunochemical and cytochemical assays. Concerning BJe, morin and PMD tested in vivo, each of them demonstrated to be capable of perturbing colon carcinogenesis as suggested by the reduction in MDFs number and size, possibly through a combination of proapoptotic and pro-inflammatory actions as suggested by in vitro experiments and also in an ex-vivo model of adenoma (for PMD study) from Pirc rats (Tortora et al. 2018). The novelty of these studies was represented by either the use of non-canonical sources of natural compounds (fruit by-products) or by the possibility to target an oncoprotein (LMW-PTP, low molecular weight phospho-tyrosine phosphatase) consequently enhancing the therapeutic response (study on morin). These data support the idea that a combination of natural compounds acting synergistically with each other, and possibly with drugs, can represent a promising secondary and tertiary chemopreventive strategy. The data obtained on colon fibroblasts mutated in Apc suggest that this mutation determines the development of a proinflammatory phenotype in this tissue microenvironment component, which might be involved in the creation of a protumorigenic environment favoring micro and macro pre-neoplastic lesion development. Moreover, data obtained with the COMET assay on colon fibroblasts and NMs from one month-old Pirc and Wt rats showed a lower level of oxidative damage in Pirc compared to Wt animals: also based on previous data from this lab (Femia et al. 2015), we speculate that Apc mutation could account for a selective advantage for carcinogenesis development through an increase in antioxidants defenses, as reported in the litterature (Ogasawara and Zhang 2008). Finally, we succeeded in the development of a methyl sensitive COMET assay, as proven by the reliability of the methylation changes observed after hypo- and hypermethylating stimuli in two different kinds of normal colon cell lines (epithelial and fibroblasts): this method will be used in the future to address the link between Apc gene and methylation.

碩士
靜宜大學
食品營養研究所
99
Among Chlorella extracts, hot-water soluble extract accounts for 16-18%, the polysaccharide or glycoprotein from cellulose hydrolysate accounts for 8%, leaving the insoluble part of ~60%. This study was to isolate hot-water soluble extracts (WS) and insoluble (WIS) components of Chlorella sorokiniana with various extraction conditions, including enzymatic treatments (cellulase, chitinase and tannase), different solvents (ethyl acetate and n-hexane), and fractionation (WS > 10, 1-10 and <1 kDa). The chemical compositions (monosaccharides, phenolics, and organic acids) the inhibition effect on human colon adenocarcinoma cell (Caco-2) and stimulation on TGF-β1 secretion of human foreskin fibreskin fibroblast (Hs68) were examined on the extracts and selective fractions. The results of monosaccharide composition analysis indicated that the sugars in WS and enzyme hydrolysis fraction are mainly glucose and galactose, following by ribose and mannose. The results of polyphenolic compounds showed that the direct solvent extracts (EaS and HS) and the solvent extracts of WIS (WIS-EaS and WIS-HS) exhibited an order that lutein, HS > WIS-HS > EaS > WIS-EaS. The extraction efficiency (based on total polyphenolics yield) of n-hexane was greater than that of ethyl acetate. The tannase hydrolysate of WIS (WIS-TaS) had an significantly antitumor activity against Caco-2 cells (at 0.1 mg/mL, inhibition: 57.23% ). Mixture of TaS and gallic acid exhibited a high inhibitory effect (78.4% at 0.2 mg/mL). The WIS-EaS stimulated Hs68 proliferation (134% at 5 μg/mL) and TGF-β1 secretion, more significantly than did the WIS-HS. In conclusion, WIS-TaS exhibited an excellent inhibition activity on Caco-2 cells. Gallic acid exhibited the best cell inhibition activity against Caco-2 among the polyphenolics examined. The WIS-EaS and WIS-HS stimulated TGF-β1 secretion from Hs68, implying the potential in wound healing.

Research Doctorate - Doctor of Philosophy (PhD)
The HSP plays a pivotal role in the spatial and temporal regulation of cell proliferation and differentiation. Conversely aberrant Hh signalling is involved in Gorlin syndrome, basal cell carcinoma (the most common cancer in the world), and more than one third of all human medulloblastoma cases. In all of these cases, it is believed that deregulated Hh signalling leads to increased cell proliferation and tumour formation. Inhibition of the Hedgehog Signalling Pathway, is a recently validated anti-cancer drug target, with vismodegib (GDC-0449, Erivedge®) and sonidegib (LDE225, Odomzo®), approved by the U.S. Food and Drug Administration for treatment of early and advanced basal cell carcinomas. We developed three new scaffolds of small molecule inhibitors of the HSP. The first scaffold consisted of 11 quinolone-2-(1H)-ones developed from a sequential Ugi-Knoevenagel reaction pathway (Chapter 3). These analogues not only express their anti-hedgehog activity through the significant inhibition of Gli₂ at both gene and protein expression in SAG-activated Shh LIGHT 2 cells at 10 and 25 μM, respectively, but are able to suppress a panel of nine human HSP expressing cancer cells (GI₅₀ from 2.9 to 18.0 μM). Whilst the exact mechanism remains to be determined, it is probable the inhibition observed is occurring downstream of Smo, due to its activity in the presence of SAG, a potent Smo activator. Subsequent second and third generation analogues were developed on the quinolone-2-(1H)-one pharmacophore, which highlighted the importance of a C3-tethered indole moiety. These new scaffolds were built on tryptophan (9 analogues, Chapter 4) and benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amine derivatives (11 analogues, Chapter 4) displaying superior inhibitory activity against Gli protein expression with the best inhibitors displaying submicromolar IC₅₀ (Chapter 4). Noteworthy, active compounds from the second and third libraries displayed inhibitory activity downstream of Smo, which circumvents the resistance issues experienced by the Smo inhibitors currently in use. We discovered the fourth library of 1,3-thiazine-6-phenylimino-5-carboxylates in a multicomponent one pot synthesis (12 analogues, Chapter 5). These analogues display structural similarities to HPI-1, a non-selective Gli inhibitor, and thus may present themselves as HSP inhibitors. Current biological evaluation is going on to investigate their anti-hedgehog properties.