Tesi sul tema "Bone Diseases, Metabolic – physiopathology"

Bone diseases such as osteoporosis and rickets cause significant reduction in bone quantity and quality, leading to mechanical abnormalities. While the reduction of bone quantity can be assessed using clinical tools like DXA and pQCT, there is little quantitative knowledge of how altered bone quality in diseased bone increases fracture risk. There is a clear need to develop high-resolution diagnostic techniques to close the gap between onset of fracture relevant changes and diagnosis. Here, a functional imaging technique (in situ synchrotron X-ray imaging with micromechanics) was developed to measure alterations in fibrillar deformation mechanisms in rickets, glucocorticoid-induced osteoporosis (GIOP), and premature ageing. During applied loading, percentage shifts in Bragg peak positions arising from the meridional collagen stagger, measured from the small angle X-ray scattering (SAXS) patterns, give fibrillar level strain as a function of applied stress in real time. To link nanostructural changes to altered fracture risk and deformability, well defined animal (mouse) models created via N-ethylnitrosurea mutagenesis were used. The fibril modulus, maximum fibril strain and fibril-to-tissue strain ratio were determined, complemented by quantitative backscattered scanning electron microscopy and microcomputed tomography to measure microscale mineralisation. A significant reduction of fibril modulus and enhancement of maximum fibril strain was found in rickets and GIOP mice. A significantly larger fibril strain/tissue strain ratio was found in GIOP mice compared to wild-type mice, indicative of a lowered mechanical competence at the bone matrix level. The effects of altered in vivo muscular force distributions on the skeletal system in rickets were measured using position resolved scanning SAXS. Increase of mineral nanoplatelet alignment is observed in wild-type mice near zones of large in-vivo muscle force but not in rachitic mice. These results demonstrate the ability of synchrotron-based in situ X-ray nanomechanical imaging to identify functional alterations in nanoscale bone quality in metabolic bone diseases.

The purpose of this study was to determine whether radiologists could accurately assess osteopenia on chest plain films. Two chest radiologists evaluated lateral chest films from 100 patients (80 female and 20 male), ranging in age from 16 to 86 years, for osteopenia and its associated findings. Intra- and interobserver agreement was determined using weighted kappa statistics, and accuracy was assessed by making comparisons to bone mineral density as measured by the non-invasive gold standard of dual-energy x-ray absorptiometry (DXA). Overall, radiologists were good at identifying signs of late, but not early, disease. Intraobserver consistency was substantial for fish vertebrae (Kw1=0.638; Kw2=0.0.712) with moderate interobserver agreement (Kw=0.45). Similarly for wedged vertebrae, intraobserver consistency was substantial to moderate (Kw1=0.654; Kw2=0.533) with substantial interobserver agreement (Kw=0.622). These radiographic signs correlated with true disease as shown by high specificity values. Therefore, this study indicates that if osteopenia is suspected (i.e., there is a wedge or fish vertebra) or its associated features are seen on a CXR, it is crucial for radiologists to comment on it. The literature suggests that referring physicians do not pay attention to such findings in radiology reports. Radiologists could effect change in clinical treatment by not burying these findings in the report body, but instead putting it in the impression, along with a recommendation that the finding be followed up with DXA. Because effective interventions for women with osteoporosis exist, the results of this study will contribute to a major change in the practice of chest radiology and improve womens health by preventing the devastating disability associated with osteoporosis.

Made available in DSpace on 2016-02-05T18:30:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-07-28. Added 1 bitstream(s) on 2016-02-05T18:34:16Z : No. of bitstreams: 1 000857773.pdf: 1062070 bytes, checksum: 71a62107765cf558c1418c16cda5314d (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O objetivo desse estudo foi analisar se há diferenças no efeito do desuso e da deficiência de estrógeno sobre o tecido ósseo trabecular e cortical, e se estes efeitos influenciam na qualidade do tecido ósseo aumentando sua fragilidade. Para este estudo, 30 ratas Wistar com 19 semanas de idade foram distribuídas nos grupos: controle (CON), descarregamento dos membros posteriores (HLU) e ovariectomizado (OVX). As análises da densidade óssea in vivo (DXA) dos fêmures e tíbias e as dosagens plasmáticas de cálcio, fósforo, fosfatase alcalina, TRAP (espectrofotometria) e E2 (ELISA) foram realizadas no início e fim do experimento, com 19 e 27 semanas de idade respectivamente. Na 27a semana, os fêmures e as tíbias foram desarticulados e armazenados para avaliar a microestrutura do osso trabecular e cortical (microtomografia computadorizada), além das propriedades biomecânicas do colo femoral e da diáfise femoral e tibial (ensaio mecânico). O grupo HLU apresentou diminuição na concentração plasmática de cálcio e atividade da fosfatase alcalina total, diminuição na DMOa do fêmur com aumento na porosidade cortical e diminuição na resistência óssea, entretanto, não foram observadas estas alterações na tíbia. O grupo OVX apresentou diminuição nas concentrações plasmáticas de cálcio, diminuição da DMOa do fêmur, deterioração trabecular no fêmur e na tíbia, com maior deterioração nas trabéculas ósseas tibiais, sem alteração na resistência óssea em ambos os ossos. Esses resultados demonstram que apesar do grupo HLU e OVX apresentassem alterações na densidade mineral óssea e microarquitetura óssea, podemos concluir que o desuso determinou maior perda no tecido cortical e na resistência óssea em relação à deficiência de estrógeno. Portanto, as análises da estrutura do tecido cortical, como a porosidade cortical, podem ser preponderantes para prever o risco de fratura
The objective of this study was to analyze whether there are differences in the effect of disuse and estrogen deficiency on trabecular and cortical bone tissue, and whether these effects influence the quality of bone tissue increasing its fragility. For this study, 30 Wistar rats with 19 weeks old, were divided into groups: control (CON), hindlimb unloading (HLU) and ovariectomy (OVX). In vivo analysis of bone density (DXA) from femurs and tibias and plasma levels of calcium, phosphorus, alkaline phosphatase, TRAP (spectrophotometry) and E2 (ELISA) were performed at the beginning and end of the experiment, and 19 age 27 weeks, respectively. In the 27th week, the femur and tibia were disjointed and stored to assess the microstructure of trabecular and cortical bone (microcomputed tomography) and biomechanical properties of the femoral neck and femoral shaft and tibial (mechanical tests). The HLU group showed a decrease in plasma calcium concentration and total alkaline phosphatase activity, decreased femoral BMAD with increased cortical porosity and decrease in bone strength, however, there were no such changes in the tibia. The OVX group showed a decrease in plasma concentrations of calcium, decreased femoral BMAD, trabecular deterioration in the femur and tibia, with further deterioration in the tibial trabecular bone, with no change in bone strength in both bones. These results demonstrate that although the HLU and OVX group showed changes in bone mineral density and bone microarchitecture, we can conclude that the in disuse determined higher cortical tissue loss and bone strength relative to estrogen deficiency. Therefore, the analysis of the cortical tissue structure, such as cortical porosity can be prevalent to predict fracture risk

Orientador: Mário Jefferson Quirino Louzada
Banca: William Dias Belangero
Banca: José Carlos Camargo Filho
Resumo: O objetivo desse estudo foi analisar se há diferenças no efeito do desuso e da deficiência de estrógeno sobre o tecido ósseo trabecular e cortical, e se estes efeitos influenciam na qualidade do tecido ósseo aumentando sua fragilidade. Para este estudo, 30 ratas Wistar com 19 semanas de idade foram distribuídas nos grupos: controle (CON), descarregamento dos membros posteriores (HLU) e ovariectomizado (OVX). As análises da densidade óssea in vivo (DXA) dos fêmures e tíbias e as dosagens plasmáticas de cálcio, fósforo, fosfatase alcalina, TRAP (espectrofotometria) e E2 (ELISA) foram realizadas no início e fim do experimento, com 19 e 27 semanas de idade respectivamente. Na 27a semana, os fêmures e as tíbias foram desarticulados e armazenados para avaliar a microestrutura do osso trabecular e cortical (microtomografia computadorizada), além das propriedades biomecânicas do colo femoral e da diáfise femoral e tibial (ensaio mecânico). O grupo HLU apresentou diminuição na concentração plasmática de cálcio e atividade da fosfatase alcalina total, diminuição na DMOa do fêmur com aumento na porosidade cortical e diminuição na resistência óssea, entretanto, não foram observadas estas alterações na tíbia. O grupo OVX apresentou diminuição nas concentrações plasmáticas de cálcio, diminuição da DMOa do fêmur, deterioração trabecular no fêmur e na tíbia, com maior deterioração nas trabéculas ósseas tibiais, sem alteração na resistência óssea em ambos os ossos. Esses resultados demonstram que apesar do grupo HLU e OVX apresentassem alterações na densidade mineral óssea e microarquitetura óssea, podemos concluir que o desuso determinou maior perda no tecido cortical e na resistência óssea em relação à deficiência de estrógeno. Portanto, as análises da estrutura do tecido cortical, como a porosidade cortical, podem ser preponderantes para prever o risco de fratura
Abstract: The objective of this study was to analyze whether there are differences in the effect of disuse and estrogen deficiency on trabecular and cortical bone tissue, and whether these effects influence the quality of bone tissue increasing its fragility. For this study, 30 Wistar rats with 19 weeks old, were divided into groups: control (CON), hindlimb unloading (HLU) and ovariectomy (OVX). In vivo analysis of bone density (DXA) from femurs and tibias and plasma levels of calcium, phosphorus, alkaline phosphatase, TRAP (spectrophotometry) and E2 (ELISA) were performed at the beginning and end of the experiment, and 19 age 27 weeks, respectively. In the 27th week, the femur and tibia were disjointed and stored to assess the microstructure of trabecular and cortical bone (microcomputed tomography) and biomechanical properties of the femoral neck and femoral shaft and tibial (mechanical tests). The HLU group showed a decrease in plasma calcium concentration and total alkaline phosphatase activity, decreased femoral BMAD with increased cortical porosity and decrease in bone strength, however, there were no such changes in the tibia. The OVX group showed a decrease in plasma concentrations of calcium, decreased femoral BMAD, trabecular deterioration in the femur and tibia, with further deterioration in the tibial trabecular bone, with no change in bone strength in both bones. These results demonstrate that although the HLU and OVX group showed changes in bone mineral density and bone microarchitecture, we can conclude that the in disuse determined higher cortical tissue loss and bone strength relative to estrogen deficiency. Therefore, the analysis of the cortical tissue structure, such as cortical porosity can be prevalent to predict fracture risk
Mestre

Submitted by FERNANDA FERNANDES null (fernanda.fernandes.ata@gmail.com) on 2016-09-08T15:50:56Z No. of bitstreams: 1 DISSERTAÇÃO FERNANDA FERNANDES.pdf: 3056607 bytes, checksum: d763bb262cd01286f3a01d7fb1bcbab4 (MD5)
Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-09T13:14:26Z (GMT) No. of bitstreams: 1 fernandes_f_me_araca.pdf: 3056607 bytes, checksum: d763bb262cd01286f3a01d7fb1bcbab4 (MD5)
Made available in DSpace on 2016-09-09T13:14:26Z (GMT). No. of bitstreams: 1 fernandes_f_me_araca.pdf: 3056607 bytes, checksum: d763bb262cd01286f3a01d7fb1bcbab4 (MD5) Previous issue date: 2016-08-02
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Este estudo teve como objetivo analisar e comparar a ação da ocitocina (OT) no metabolismo ósseo de ratas Wistar cíclicas (12 meses) e acíclicas (18 meses/período de periestropausa). Os animais foram distribuídos em quatro grupos: (1) animais com 12 meses que receberam injeção com veículo NaCl (Veh/12); (2) animais com 12 meses que receberam injeção de OT (Ot / 12); (3) animais com 18 meses que receberam injeção com veículo NaCl (Veh/18); (4) animais com 18 meses que receberam injeção de OT (Ot / 18). Cada grupo foi composto por 8 animais. Os animais receberam duas injeções intraperitoneais de NaCl (0,15 M – grupo controle) ou OT (134 ug / kg – grupo tratado) com 12 horas de intervalo. Força, microarquitetura e biomarcadores ósseos [fosfatase alcalina (FAL) e fosfatase ácida resistente ao tartarato (TRAP)] foram analisados. Imunoistoquímica foi realizada para fator de transcrição relacionado com o Runt 2 (RUNX2), osterix (OSX), osteocalcina (OCN), osteopontina (OPN), proteína óssea morfogenética 2 e 4 (BMP-2/4), periostina (PER), esclerostina (ESC) e TRAP. Os animais que receberam OT demonstraram melhora significante na dosagem plasmática: aumento na FAL dos animais de 12 meses (p < 0,0001) e 18 meses (p = 0,0138); diminuição na TRAP dos animais de Ot / 12 (p = 0,0465) e Ot / 18 (p = 0,0045). Houve melhora nos parâmetros biomecânicos: força máxima (N) do grupo Ot / 18 (p = 0,0003); rigidez óssea (x103N/mm) do grupo Ot / 12 (p = 0,0223) e Ot / 18 (p = 0,0145); microarquitetura óssea cortical do grupo Ot / 18 para Ct.Ar (mm2 ) (p = 0,0416) e Ct.Po (%) (p = 0,0102); microarquitetura óssea trabecular para Tb.N (1/mm) (p = 0,0016) e Tb.Sp (p = 0,00010); todos os grupos foram comparados ao seus respectivos controles (Veh/12; Veh/18). Em resumo, os resultados demonstraram que a administração de OT foi eficaz para prevenir a perda de massa óssea em ratas Wistar velhas com hipoestrogenismo, reforçando este agente anabólico como forte alternativa terapêutica para prevenção e tratamento da osteoporose (OP), reduzindo os índices da doença e fraturas osteoporóticas.
This study aimed to analyze and compare the acting of oxytocin (OT) on bone metabolism of cyclic (12 months) and acyclic Wistar rats (18 months/peri-estropause period). Animals were allocated to four groups: (1) animals with 12 months that received vehicle injection NaCl (Veh/12); (2) animals with 12 months that received OT injection (Ot / 12); (3) animals with 18 months that received vehicle (Veh/18) and (4) animals with 18 months that received OT injection (Ot / 18). Eight animals composed each group. The animals received two intraperitoneal injections of NaCl (0.15 M - control group) or OT (134 ug / kg - treated groups) with 12 hours apart. Bone strength, microarchitecture, and biomarkers [alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP)] were assessed. Immunohistochemistry was performed for runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN), osteopontin (OPN), bone morphogenetic protein 2 and 4 (BMP- 2/4), periostin (PER), sclerostin (ESC) and TRAP. Animals that received OT showed significant improvements at plasma assay: increase in the ALP from the animals with 12 months (p < 0.0001) and 18 months (p = 0.0138); decrease in the TRAP from the Ot / 12 (p = 0.0465) and Ot / 18 (p = 0.0045). There was improvements at biomechanical parameters: maximum load (N) from the Ot / 18 (p = 0.0003); bone stiffness (x103N/mm) from the Ot / 12 (p = 0.0223) and Ot / 18 (p = 0.0145); cortical bone microarchitecture from the Ot / 18 to Ct.Ar (mm2 ) (p = 0.0416) and Ct.Po (%) (p = 0.0102); trabecular bone microarchitecture to Tb.N (1/mm) (p = 0.0016) and Tb.Sp. (p = 0.00010); all groups compared to its respective controls (Veh/12; Veh/18). In summary, the results showed the OT administration was effective to prevent the bone loss in old Wistar rats with hypoestrogenism, reforcing this anabolic agent as powerful therapeutic alternative to prevention and treatment for osteoporosis (OP), to reduce the rates of disease and osteoporotic fractures.
CNPq: 133834/2014-0

Orientador: Mário Jefferson Quirino Louzada
Banca: Ivania Garavella
Banca: Leda Maria Pescinini Salzedas
Resumo: A literatura apresenta que a resposta de reparo ósseo pode ser acentuada pela estimulação física, mecânica ou eletromagnética. Há evidências de que o ultra-som - US - de baixa potência pode acelerar a regeneração óssea. Este trabalho objetivou verificar o efeito do US no defeito ósseo, criado experimentalmente, em tíbias de ratos sob ausência de carga (suspenso pela cauda) por meio de análise densitométrica e biomecânica. Trinta Rattus novergicus albinus, Wistar, adultos, divididos em 3 grupos: G1 (n=10), não suspenso - experimento de 15 dias; G2 (n=10), suspenso pela cauda - experimento de 15 dias e, G3 (n=10), suspensos pela cauda, experimento de 36 dias. Os animais foram submetidos à osteotomia em ambas as tíbias e à aplicação do US (freqüência de 1,5 MHz, ciclo 1:4, 30mW/cm2) na direita (12 sessões de 20 minutos). O G3 somente foi osteotomizado após 21º dia de suspensão. Para análises densitométrica utilizou-se densitômetro DPXLunar ™, sistema digital Digora e o programa computacional Image J ; para ensaio mecânico usou máquina universal de ensaio EMIC . Os resultados do Conteúdo Mineral Ósseo (g), Área (cm²), Densidade Mineral Óssea (g/cm²) e da Densidade Óssea (mmAl) observadas nas tíbias, assim como a Força Máxima (N) e Rigidez (x103N/m) não demonstraram diferenças significantes (tratadas versus controle de cada grupo), possivelmente pelo menor tempo de tratamento com relação aos trabalhos encontrados na literatura. Concluindo que o Ultra-Som de baixa potência não acelerou o processo de consolidação óssea.
Abstract: Literature shows that bone repair response can be accented by physical, mechanic or electromagnetic stimulation. There are evidences that low power ultrasound - US - can speed up bone regeneration. This work aimed at determining the effect of US in bone defects, experimentally created, in tibia from rats under load absence (suspended by the tail) by densitometric analysis and biomechanics. Thirty Rattus novergicus albinus, Wistar, adult, divided in 3 groups: G1 (n=10), not suspended - a 15 day experiment; G2 (n=10), suspended by the tail - a 15 day experiment and, G3 (n=10), suspended by the tail - a 36 day experiment. , The animals have been submitted to the osteotomy in both tibias and to the US application (1,5 MHz frequency, cycle 1:4, 30mW/cm2), on the right (twelve sessions of 20 minutes). G3 was only osteotomized after the 21st day of suspension. DPX-Lunar™ densitometer, Digora digital system and Image J computer program were used for densitometrical analysis; for the mechanical assay, the universal machine of EMIC assay was used. The results for Bone Mineral Content (g), Area (cm²), Bone Mineral Density (g/cm²) and Bone Density (mmAl) observed in tibias, as well as Maximum Power (N), and Rigidity (x103N/m) did not show any significant differences (treated versus control of each group), possibly due to shorter treatment time as regards the studies found in literature. Concluding that the low power ultrasound not accelerated the process of consolidating bone.
Mestre

Made available in DSpace on 2015-09-17T15:24:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-10-29. Added 1 bitstream(s) on 2015-09-17T15:47:43Z : No. of bitstreams: 1 000848151.pdf: 1207361 bytes, checksum: 2b8b50bc45f9ec60b8daf94c7b8fb5dc (MD5)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Neste estudo foram avaliados os efeitos que a obesidade, induzida pelo consumo de sacarose, proporciona no tecido ósseo de ratos Wistar machos idosos, em duas situações opostas: em sedentarismo extremo e com realização de exercícios de força. O sedentarismo foi induzido pela suspensão pela cauda, simulando situações de pouca ou nenhuma carga nos membros pélvicos e os exercícios foram realizados em escada adaptada. Foram estudados 54 ratos machos com 16 meses de idade. Ao completarem 13 meses de idade, 27 ratos foram aleatoriamente selecionados para receberem dieta rica em sacarose durante 3 meses. Foram realizadas análises com o densitômetro DXA para a mensuração do conteúdo mineral ósseo, área e densidade mineral óssea nos fêmures e tíbias; Ensaios mecânicos de compressão da cabeça do fêmur, compressão da tíbia e compressão do osso cortical e análises imunoistoquímica com marcações para OCN e TRAP. Os resultados mostraram que os animais obesos que realizaram exercícios obtiveram melhora significativa no CMO dos fêmures, ao passo que os animais não obesos que realizaram exercícios obtiveram melhora no CMO dos fêmures e tíbias, DMOA dos fêmures e tíbias, e na força máxima admitida das tíbias. Os animais suspensos obtiveram uma perda significativa na qualidade óssea em todos os parâmetros analisados, com exceção da área. Baseado nos achados deste estudo, conclui-se que a falta de uso de um segmento ósseo, em Rattus novergicus albinus em idade avançada, tanto obesos quanto não obesos, leva a um rápido enfraquecimento de todos os parâmetros ósseos. Exercícios produzem moderado efeito positivo nos animais não obesos e baixo efeito positivo nos animais obesos.
In this study was evaluated the effects that obesity induced by sucrose intake provides in bone tissue of aged male Wistar rats in two opposite situations: in extreme inactivity, and performing strength exercises. Physical inactivity was induced by tail suspension, simulating situations of little or no load on the hindlimbs and the exercises were performed on adapted ladder. 54 male rats with 16 months old were studied. Upon completion 13 months old, 27 rats were randomly selected to receive sucrose-rich diet for 3 months. Analyzes were made: with densitometer to measure bone mineral content, bone mineral density and area in the femurs and tibias; Mechanical tests of compression of the femoral head, tibial compression and compression of the cortical bone and Immunohistochemical analysis with markings for OCN and TRAP. The results showed obese animals that performed exercises had significant improvement (p<0,05) in BMC of femurs, while non-obese animals that performed exercises showed improvement in BMC of femurs and tibias, ABMD of femurs and tibias, and the maximum allowed force of tibias. The suspended animals had a significant bone quality loss (p<0,05) in all parameters analyzed, except for the area. Based on our findings, we can conclude that lack of use of a bone segment in Rattus norvegicus albinus in old age, both obese and non-obese, leads to a rapid weakening of all bone parameters. Exercises provides moderate positive effect in non-obese animals and low positive effect in obese animals.

Orientador: Mário Jefferson Quirino Louzada
Banca: José Carlos Silva Camargo Filho
Banca: Roberta Okamoto
Resumo: Neste estudo foram avaliados os efeitos que a obesidade, induzida pelo consumo de sacarose, proporciona no tecido ósseo de ratos Wistar machos idosos, em duas situações opostas: em sedentarismo extremo e com realização de exercícios de força. O sedentarismo foi induzido pela suspensão pela cauda, simulando situações de pouca ou nenhuma carga nos membros pélvicos e os exercícios foram realizados em escada adaptada. Foram estudados 54 ratos machos com 16 meses de idade. Ao completarem 13 meses de idade, 27 ratos foram aleatoriamente selecionados para receberem dieta rica em sacarose durante 3 meses. Foram realizadas análises com o densitômetro DXA para a mensuração do conteúdo mineral ósseo, área e densidade mineral óssea nos fêmures e tíbias; Ensaios mecânicos de compressão da cabeça do fêmur, compressão da tíbia e compressão do osso cortical e análises imunoistoquímica com marcações para OCN e TRAP. Os resultados mostraram que os animais obesos que realizaram exercícios obtiveram melhora significativa no CMO dos fêmures, ao passo que os animais não obesos que realizaram exercícios obtiveram melhora no CMO dos fêmures e tíbias, DMOA dos fêmures e tíbias, e na força máxima admitida das tíbias. Os animais suspensos obtiveram uma perda significativa na qualidade óssea em todos os parâmetros analisados, com exceção da área. Baseado nos achados deste estudo, conclui-se que a falta de uso de um segmento ósseo, em Rattus novergicus albinus em idade avançada, tanto obesos quanto não obesos, leva a um rápido enfraquecimento de todos os parâmetros ósseos. Exercícios produzem moderado efeito positivo nos animais não obesos e baixo efeito positivo nos animais obesos.
Abstract: In this study was evaluated the effects that obesity induced by sucrose intake provides in bone tissue of aged male Wistar rats in two opposite situations: in extreme inactivity, and performing strength exercises. Physical inactivity was induced by tail suspension, simulating situations of little or no load on the hindlimbs and the exercises were performed on adapted ladder. 54 male rats with 16 months old were studied. Upon completion 13 months old, 27 rats were randomly selected to receive sucrose-rich diet for 3 months. Analyzes were made: with densitometer to measure bone mineral content, bone mineral density and area in the femurs and tibias; Mechanical tests of compression of the femoral head, tibial compression and compression of the cortical bone and Immunohistochemical analysis with markings for OCN and TRAP. The results showed obese animals that performed exercises had significant improvement (p<0,05) in BMC of femurs, while non-obese animals that performed exercises showed improvement in BMC of femurs and tibias, ABMD of femurs and tibias, and the maximum allowed force of tibias. The suspended animals had a significant bone quality loss (p<0,05) in all parameters analyzed, except for the area. Based on our findings, we can conclude that lack of use of a bone segment in Rattus norvegicus albinus in old age, both obese and non-obese, leads to a rapid weakening of all bone parameters. Exercises provides moderate positive effect in non-obese animals and low positive effect in obese animals.
Mestre

Orientador: Anderson Marliere Navarro
Banca: Rebeca Antunes Beraldo
Banca: Vivian Marques Miguel Suen
Banca: Elen Almeida Romão
Banca: Rodrigo de Carvalho Santana
Resumo: A hepatite B é um importante problema de saúde pública mundial e se associa a consideráveis taxas de mortalidade. As medicações de uso oral para o tratamento da hepatite B, denominados em conjunto de análogos de nucleosídeo/nucleotídeo, são de uso prolongado e apresentam potenciais efeitos colaterais, como a redução na densidade mineral óssea que está associada ao uso do tenofovir. Objetivo: avaliar os efeitos do tenofovir, em comparação com outros análogos de nucleosídeo/nucleotídeo (entecavir e lamivudina), sobre a densidade mineral óssea de um grupo de pacientes com hepatite B crônica. Materiais e Métodos: foi realizado um estudo observacional do tipo transversal com 81 pacientes adultos, com Hepatite B crônica atendidos no Hospital das Clínicas de Ribeirão Preto - USP nos quais realizou-se os exames de densitometria óssea (dual energy x-ray absorptiometry-DXA), análises bioquímicas de osteocalcina, deoxipiridinolina, vitamina D, paratormônio, IGF-1, TSH, testosterona, estradiol, FSH, transaminases, ureia, creatinina, cálcio total, fósforo sérico e urinário, magnésio, FGF-23 e medidas antropométricas (peso, altura e índice massa corporal). Os participantes, de ambos os sexos, foram subdivididos segundo o uso ou não de antirretrovirais sendo: Grupo 1: 27 pacientes portadores inativos do vírus sem uso de medicamentos; Grupo 2: 27 pacientes em uso de Tenofovir; Grupo 3: 27 pacientes em uso de Lamivudina ou Entecavir. Resultados: não houve diferença entre a idade média e índic... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Hepatitis B is one the most important public health problem worldwide and is associated with considerably high mortality rates. Oral medications, generally nucleoside/nucleotide analogs such as tenofovir, have been used as long-term therapy and have possible side effects such as the reduction in bone mineral density associated with their use. Objective: to evaluate the effects of tenofovir, compared with those of other nucleoside/nucleotide analogs (entecavir and lamivudine), on the bone mineral density of hepatitis B patients. Materials and Methods: a cross-sectional study was conducted with 81 adult patients with chronic hepatitis B treated. The average age of the participants was 42 years. Dual-energy x-ray absorptiometry (DXA) was performed to assess bone mineral density. Biochemical analyses were performed for osteocalcin, deoxypyridinoline, parathyroid hormone, vitamin D, IGF-1, TSH, testosterone, estradiol, FSH, transaminases, urea, creatinine, calcium, serum and urinary phosphorus, magnesium, FGF-23 and anthropometric measures of weight, height, and body mass index were performed. Participants, both gender, were divided according to the use of antiretrovirals: Group 1: 27 inactive virus carriers without medication; Group 2: 27 patients using tenofovir; Group 3: 27 patients using lamivudine or entecavir. Results: We did not find differences in mean age, body mass index, lean and fat mass between patients in both groups (p>0.05). DXA readings diagnosed osteopenia in the... (Complete abstract click electronic access below)
Doutor

INTRODUÇÃO: A obesidade é uma doença crônica com crescimento alarmante no mundo todo. Atualmente, o tratamento cirúrgico, especialmente o Bypass Gástrico em Y de Roux (BGYR), tem se mostrado como a forma mais eficiente para perda de peso e sua manutenção a longo prazo. Contudo, com a formação do neo-estômago e a mudança na conformidade intestinal, há alterações significantes das muitas propriedades físicas e funcionais desses órgãos que levam à deficiência de nutrientes, inclusive de cálcio. Com isso, podem ocorrer modificações no metabolismo ósseo e, conseqüentemente, na estrutura óssea. OBJETIVOS: Avaliar a ingestão de cálcio, as alterações no metabolismo ósseo e mineral; e a ocorrência de osteopenia e osteoporose em mulheres que se submeteram ao BGYR há oito anos. MÉTODO: Neste estudo transversal, foram estudadas 30 mulheres que se submeteram ao BGYR no período de outubro de 1995 a janeiro de 1999, no Hospital das Clínicas da Faculdade de Medicina da USP. Para avaliação da ingestão de cálcio, utilizamos o recordatório de 3 dias (R3D) e o questionário de freqüência alimentar (QFA). Também foram realizados exames laboratoriais referentes ao metabolismo ósseo e mineral e densitometria óssea do seguimento L1-L4, colo femoral (CF) e fêmur proximal (FP). RESULTADOS: Em média, o consumo de cálcio foi de 525,5 ± 250,7 mg/dia pelo R3D e de 542,2 ± 195,6 mg/dia pelo QFA. Houve uma relação estatisticamente significativa entre a ingestão de cálcio por esses dois métodos (p<0,001). Não houve alteração nas determinações de cálcio total e ionizado, magnésio, fósforo e CTX. Os níveis de PTH, Fosfatase alcalina fração óssea (BSAP) e osteocalcina estavam elevados em 53%, 57% e 20% das mulheres, respectivamente; 90% apresentavam deficiência de 25 (OH) vitamina D (40% leve e 50% moderada), e em 70% a calciúria estava abaixo dos valores normais. Observou-se uma correlação positiva entre 25 (OH) vitamina D e a calciúria (p<0,04) e negativa entre 25 (OH) vitamina D e PTH (p<0,017). Com relação à densidade mineral óssea, 13% das mulheres foram diagnosticadas com osteoporose com relação ao CF e FP; 67%, 40% e 27% apresentavam osteopenia em L1-L4, CF e FP, respectivamente. CONCLUSÃO: Na maioria das mulheres estudadas verificou-se um consumo de cálcio cerca de 50% abaixo da recomendação diária para esta faixa etária. Observou-se também, uma deficiência de 25 (OH) vitamina D e elevação de PTH e BSAP. Além disso, houve uma ocorrência de osteopenia superior à esperada indicando que alterações no metabolismo ósseo são provavelmente uma complicação do BGYR. Mais estudos são necessários para definir uma rotina de suplementação de cálcio e vitamina D, e também para a prevenção das alterações ósseas.
INTRODUTION: Obesity is a chronic disease that rises rapidly around the world. Nowadays bariatric surgical procedures, especially Roux-en-Y Gastric Bypass (RYGB) has been shown the most efficient way to lose weight and maintain the weight loss for a long time. However, with the neo-stomach and the modification of intestinal anatomy by the surgery there are significant changes on physiological properties of these organs that lead to a nutrient deficiency, including calcium. Thus, bone metabolism changes may occur leading to a metabolic bone disease. OBJECTIVES: To evaluate calcium intake, bone and mineral metabolism changes and the prevalence of metabolic bone disease in women who were submitted to RYGB after eight years. METHOD: we studied 30 women who were submitted to RYGB during the period between October of 1995 and January of 1999 at Clinical Hospital of Medicine School of São Paulo University. To access calcium intake we used a 3 day dietary recall (3DR) and food frequency questionnaire (FFQ). Laboratory tests of bone metabolism and bone mass density of L1-L4, femoral neck (FN) and proximal femur (PF) were also accessed. RESULTS: calcium intake was 525,5 ± 250,7 mg/day according 3RD and 542,2 ± 195,6 mg/day according FFQ. There was a significantly relation between both methods (p<0,001). Total and ionic calcium, magnesium, phosphorus and CTX were not altered. PTH, bone specific alkaline phosphatase (BSAP) and osteocalcin levels were elevated respectively in 53%, 57% and 20% of women. 90% presented 25 (OH) vitamin D deficiency (40% mild and 50% moderate) and 70% had low urinary calcium. Was observed a positive correlation between 25 (OH) vitamin D and urinary calcium (p<0,04); and a negative correlation between 25 (OH) vitamin D and PTH (p<0,017). 13% of women had osteoporosis in FN and PF; 67%, 40% and 27% had metabolic bone disease in L1-L4, FN and PF respectively. CONCLUSION: Most studied women had a low calcium intake, about 50% of daily recommendation. We also noticed a 25 (OH) vitamin D deficiency and elevated levels of PTH and BSAP. Besides, there was a high prevalence of metabolic bone disease than expected, suggesting that this could be a complication of this surgery. Further studies are needed to define a supplementation routine of calcium and vitamin D to prevent bone metabolic diseases in these patients.

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-08Bitstream added on 2014-06-13T20:14:18Z : No. of bitstreams: 1 apolinario_jc_me_araca.pdf: 190197 bytes, checksum: 1aba6bb35b316b811a8b671145feab29 (MD5)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A literatura apresenta que a resposta de reparo ósseo pode ser acentuada pela estimulação física, mecânica ou eletromagnética. Há evidências de que o ultra-som – US – de baixa potência pode acelerar a regeneração óssea. Este trabalho objetivou verificar o efeito do US no defeito ósseo, criado experimentalmente, em tíbias de ratos sob ausência de carga (suspenso pela cauda) por meio de análise densitométrica e biomecânica. Trinta Rattus novergicus albinus, Wistar, adultos, divididos em 3 grupos: G1 (n=10), não suspenso - experimento de 15 dias; G2 (n=10), suspenso pela cauda - experimento de 15 dias e, G3 (n=10), suspensos pela cauda, experimento de 36 dias. Os animais foram submetidos à osteotomia em ambas as tíbias e à aplicação do US (freqüência de 1,5 MHz, ciclo 1:4, 30mW/cm2) na direita (12 sessões de 20 minutos). O G3 somente foi osteotomizado após 21º dia de suspensão. Para análises densitométrica utilizou-se densitômetro DPXLunar ™, sistema digital Digora e o programa computacional Image J ; para ensaio mecânico usou máquina universal de ensaio EMIC . Os resultados do Conteúdo Mineral Ósseo (g), Área (cm²), Densidade Mineral Óssea (g/cm²) e da Densidade Óssea (mmAl) observadas nas tíbias, assim como a Força Máxima (N) e Rigidez (x103N/m) não demonstraram diferenças significantes (tratadas versus controle de cada grupo), possivelmente pelo menor tempo de tratamento com relação aos trabalhos encontrados na literatura. Concluindo que o Ultra-Som de baixa potência não acelerou o processo de consolidação óssea.
Literature shows that bone repair response can be accented by physical, mechanic or electromagnetic stimulation. There are evidences that low power ultrasound – US - can speed up bone regeneration. This work aimed at determining the effect of US in bone defects, experimentally created, in tibia from rats under load absence (suspended by the tail) by densitometric analysis and biomechanics. Thirty Rattus novergicus albinus, Wistar, adult, divided in 3 groups: G1 (n=10), not suspended – a 15 day experiment; G2 (n=10), suspended by the tail – a 15 day experiment and, G3 (n=10), suspended by the tail – a 36 day experiment. , The animals have been submitted to the osteotomy in both tibias and to the US application (1,5 MHz frequency, cycle 1:4, 30mW/cm2), on the right (twelve sessions of 20 minutes). G3 was only osteotomized after the 21st day of suspension. DPX-Lunar™ densitometer, Digora digital system and Image J computer program were used for densitometrical analysis; for the mechanical assay, the universal machine of EMIC assay was used. The results for Bone Mineral Content (g), Area (cm²), Bone Mineral Density (g/cm²) and Bone Density (mmAl) observed in tibias, as well as Maximum Power (N), and Rigidity (x103N/m) did not show any significant differences (treated versus control of each group), possibly due to shorter treatment time as regards the studies found in literature. Concluding that the low power ultrasound not accelerated the process of consolidating bone.

As doenças renais crônicas (DRC) evoluem com distúrbios na homeostase do cálcio e do fósforo, diminuição na produção de vitamina D e aumento na secreção de PTH. Osteodistrofia renal (OR) é o termo usado para definir as alterações ósseas dos pacientes com DRC e classifica-se em doença de alta remodelação representada pela osteíte fibrosa (OF) e doença mista (DM); e de baixa remodelação representada pela osteomalácia (OM) e pela doença adinâmica (DOA). Pacientes com DRC apresentam elevada incidência de fraturas e recentemente demonstrou-se que a hiperfosfatemia leva a diminuição do volume ósseo. Estudamos o efeito isolado do fósforo no tecido ósseo de animais com insuficiência renal mantidos com infusão fixa de PTH variando o conteúdo de fósforo na dieta. Cinqüenta e cinco ratos Wistar foram submetidos à paratireoidectomia (PTX) e nefrectomia (Nx) com reposição de PTH em diferentes concentrações ou foram sham operados e recebiam infusão de veículo. Todos os animais receberam a mesma dieta variando apenas a concentração de P (pobre em P (pP): 0,2% e rico em P (rP):1,2%). Dividimos os grupos em: Sham (N=8); Sham-pP (N=8); Sham-rP (N=7); NxPTHn-pP (N=8); NxPTHn-rP (N=8); NxPTHe-pP (N=9); NxPTHe-rP (N=7). Após 2 meses, realizamos análises bioquímicas e histomorfometria do fêmur proximal. Os animais que ingeriram dieta rica em fósforo apresentaram hiperfosfatemia assim como menor valor de cálcio sérico. A reposição de PTH foi efetiva e proporcional às concentrações infundidas. A histomorfometria óssea mostrou que os ratos que ingeriram dieta rica em fósforo independente da uremia tinham diminuição do volume ósseo (BV/TV), e que este efeito foi amenizado pela reposição do PTH em concentrações elevadas. Nossos resultados demonstram que o fósforo é deletério para o tecido ósseo e que na uremia são necessários níveis mais elevados de PTH para manter a integridade óssea.
Chronic kidney disease (CKD) involves disturbances in calcium and phosphorus metabolism, reduced vitamin D production and increased parathormone (PTH) secretion. Renal osteodistrophy (RO) is a term used to define bone disease complications of patients with CKD, and is classified in high turnover disease represented by osteitis fibrosa (OF) and mixed bone disease; and low turnover disease represented by osteomalacia (OM) and adynamic bone disease (ABD). It is already known that patients with CKD have high incidence of bone fractures, and it has been demonstrated that hyperphosphatemia results in to decreased trabecular bone volume (BV/TV). We evaluated the effect of phosphorus (P) in rats? bone tissue submitted to experimental uremia that received continuous infusion of 1-34 rat PTH in physiologic or five times the normal values. Fifty five Wistar rats were submitted to parathyroidectomy (PTX), nephrectomy (Nx) and received PTH in different concentrations or some were PTX and NX controls (Sham) that received only vehicle. Rats received identical diets, excepted for the P content which was different according to the group [Low P (LP): 0,2% and high P (HP): 1,2%]. Groups were divided as follow: Sham (N=8), Sham LP (N=8), Sham-HP (N=7), NxPTHn-LP (N=8), NxPTHn-HP (N=8), NxPTHh-LP (N=9), NxPTHh-HP (N=7). After two months, animals were sacrificed and biochemical and bone histomorphometry were performed. Rats who received high P diet developed hyperphosphatemia and hypocalcemia. PTH replacement was effective and in accordance with infusion concentration. Bone histomorphometric analysis showed that HP rats presented low trabecular bone volume (BV/TV) independently of the uremia. BV/TV decreased slightly in the group where PTH continuous infusion was five times the physiologic values. Our results demonstrated that P has a deleterious action on bone tissue and in uremia it is necessary high levels of PTH to maintain bone integrity.

Orientador: Anderson Marliere Navarro
Banca: Alcyone Artioli Machado
Banca: Telma Maria Braga Costa
Banca: Paulo Inácio da Costa
Banca: Alceu Afonso Jordão Júnior
Resumo: A história natural da infecção pelo HIV e a terapia antirretroviral (TARV) já estão bem esclarecidos, por proporcionarem aumento da contagem das células T CD4+, redução da viremia e diminuição de riscos para doenças oportunistas. Apesar da eficácia do tratamento antirretroviral, evidenciam-se os efeitos sistêmicos da inflamação mediados pela infecção crônica do HIV e a toxicidade dos antirretrovirais que contribuem para o aumento de riscos de complicações metabólicas. Nós hipotizamos que pacientes HIV positivos em uso ou não da terapia antirretroviral apresentam níveis aumentados nos marcadores inflamatórios, e isto afeta o metabolismo ósseo que leva à perda óssea, sendo mais acometidos os pacientes com maior tempo de exposição ao vírus e ao tratamento antirretroviral. Objetivo: Investigar a influência de citocinas pró-inflamatórias no metabolismo ósseo em pacientes HIV positivos crônicos em uso ou não da terapia antirretroviral. Métodos: Trata-se de um estudo do tipo transversal com 50 homens adultos HIV positivos, em tratamento ou não com drogas antirretrovirais. Os participantes foram subdivididos segundo o uso ou não da TARV, sendo grupo controle (GC): 10 participantes virgens de tratamento; grupo G<2 anos de TARV: 20 participantes abaixo de dois anos de tratamento antirretroviral; grupo G>2 anos de TARV: 20 participantes acima de dois anos de tratamento antirretroviral. Foi realizado dual energy x-ray absorptiometry (DXA) para avaliar a densidade mineral óssea e a compos... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The natural history of HIV infection and antiretroviral treatment (ART) are well evidenced by increase in the CD4+ T cell count, reduction of viral replication, and reduced risk for opportunistic diseases. Despite the efficacy of ART, the current repercussions are the systemic effects of inflammation mediated by chronic HIV infection and toxicity of the antiretroviral drugs that significantly contribute to increase the risk of metabolic complications. We hypothesized that HIV-infected patients treated or not with antiretroviral therapy have increased levels of inflammatory markers that can affect bone metabolism leading to bone loss, and the patients with longer exposure to HIV and ART are more affected. Objective: Investigate the influence of pro-inflammatory cytokines in bone metabolism in patients with chronic HIV infection treated or not with antiretroviral therapy. Methods: A cross-sectional study was conducted on 50 HIV-seropositive men treated or not with ART. The participants were divided, according to the use or not of ART, into the control group (CG): 10 participants not in treatment; the G<2 years of ART group: 20 participants treated with ART for less than 2 years; and the G>2 years of ART group: 20 participants treated with ART for more than 2 years. Dual energy x-ray absorptiometry (DXA) was performed to evaluate bone mineral density and body composition... (Complete abstract click electronic access below)
Doutor

Distúrbios do metabolismo mineral e ósseo são altamente prevalentes e considerados como causa relevante da morbidade e mortalidade dos pacientes com doença renal crônica. Diversas estratégias diagnósticas e terapêuticas têm sido estudadas nesses doentes; entretanto, pouco valor é dado ao cálcio do dialisato, apesar do impacto que possa exercer sobre o balanço de cálcio durante a hemodiálise. Os fatores determinantes da transferência de cálcio durante o procedimento são ainda controversos. Nesse estudo prospectivo, avaliamos a influência da remodelação óssea sobre o balanço de cálcio em dez pacientes dialíticos em três situações consecutivas: hiperparatireoidismo grave (Pré paratireoidectomia), durante a \"síndrome de fome óssea\" (Fome óssea) imediatamente após a paratireoidectomia e após estabilização clínica (Paratireoidectomia tardia). Durante cada fase os participantes foram submetidos a três sessões randômicas de hemodiálise com diferentes concentrações de cálcio no dialisato: 2,5; 3,0 e 3,5 mEq/L. Todos os pacientes foram submetidos à biópsia óssea para análise histomorfométrica e quantificação de proteínas ósseas no início do estudo. A transferência de cálcio variou grandemente entre os pacientes em cada fase do estudo mesmo usando o mesmo cálcio no dialisato, com valores negativos de medianas no Pré paratireoidectomia e Fome óssea (-161mg e -218mg, respectivamente) e discretamente positivo no Paratireoidectomia tardio (39mg; p < 0,05 versus Pré paratireoidectomia e Fome óssea). Análise de regressão multivariada mostrou que o gradiente de cálcio entre o cálcio iônico sérico inicial e o cálcio do dialisato, a diferença entre o cálcio iônico sérico final e o inicial e a forma não carboxilada da osteocalcina foram preditores independentes da transferencia de cálcio (R2=0.48; p < 0.05). Pelo fato da remodelação óssea também influenciar os níveis séricos de cálcio iônico e suas variações durante a diálise, nesse estudo demonstramos que o esqueleto tem papel fundamental no balanço de cálcio e essas variáveis devem ser consideradas na individualização do cálcio do dialisato de nossos pacientes
Disturbances in mineral and bone metabolism are highly prevalent and are a major cause of morbidity and mortality in chronic kidney disease patients. Different diagnostic and therapeutic strategies have been studied in these patients. However, little attention is paid to the calcium concentration in the dialysate, despite the impact it could exert over calcium balance during dialysis. The variables that determine calcium transfer during hemodialysis are still controversial. In this study, we have prospectively investigated the influence of bone remodeling on calcium balance in ten dialysis patients in three consecutive situations: severe hyperparathyroidism (Pre parathyroidectomy), during \"hungry bone syndrome\" (Hungry bone) right after surgery and after stabilization of clinical status (Late parathyroidectomy). During each phase participants were submitted to 3 random hemodialysis sessions, with different dialysate calcium: 2.5, 3.0 and 3.5 mEq/L. Bone biopsy for hystomorphometric analysis and bone proteins quantification were performed in all patients at baseline. Calcium mass transfer varied widely among patients in each study phase even using the same dialysis calcium with negative median values in Pre parathyroidectomy and Hungry Bone (-161 and -218mg, respectively) and slightly positive in Late parathyroidectomy (39mg; p<0.05 versus Pre parathyroidectomy and Hungry Bone). Multiple regression analysis showed that calcium gradient between initial serum ionic calcium and the dialysate calcium, the difference between final and initial serum ionic calcium and serum undercarboxylated form of osteocalcin were independent predictors of calcium mass transfer (R2=0.48; p<0.05). As bone remodeling also influences the serum levels of ionic calcium and its variance during dialysis, in this study we have added new data by demonstrating that the skeleton plays a key role on calcium balance and these variables must be considered when individualizing calcium dialysate for our patients

A maior parte dos distúrbios metabólicos da doença renal crônica (DRC) é revertida após um transplante renal bem-sucedido. Porém, alterações do metabolismo ósseo podem permanecer e estão associadas ao aumento de fraturas, calcificação vascular, perda de enxerto e mortalidade. A expressão óssea de proteínas osteocíticas está alterada na DRC e parece contribuir negativamente para a homeostase óssea. Há relatos de aumento da expressão óssea de FGF-23 e esclerostina em crianças que receberam transplante de órgãos sólidos em comparação com voluntários normais. Entretanto, análise da expressão destas proteínas em receptores adultos ainda não foi realizada. Avaliação de biopsia óssea em 31 pacientes uma semana antes e 1 ano após o transplante renal. Realizada histomorfometria óssea e avaliação das proteínas ósseas através de imunohistoquimica, multiplex e expressão gênica. Na avaliação das biópsias antes do transplante, houve concordância entre os achados de imunohistoquimica e multiplex para esclerostina e FGF-23. Um ano após o transplante renal bem-sucedido, observamos diminuição dos níveis séricos do PTH, TRAP5b, fosfatase alcalina óssea, FGF-23, OPG e esclerostina. Apesar da diminuição da esclerostina sérica, houve aumento de seu conteúdo ósseo pela imunohistoquimica, multiplex e expressão gênica. Também foi observado aumento do conteúdo proteico e da expressão gênica da beta-catenina fosforilada, confirmando a inibição da via Wnt. Esta inibição foi acompanhada do aumento do conteúdo ósseo de RANKL e diminuição da OPG. Em relação ao FGF-23, houve concordância entre níveis séricos e conteúdo proteico, confirmando sua menor síntese pelos osteócitos, e portanto, menor nível sérico, após o transplante renal. A recuperação da função renal após o transplante é acompanhada de mudanças nas proteínas séricas e ósseas. A esclerostina óssea aumentou, apesar da diminuição do nível sérico, acompanhada de mudanças em outras proteínas que confirmam a inibição da via Wnt. Esse achado pode ajudar a desvendar a fisiopatologia da doença óssea pós transplante e guiar a busca por novas terapias
Most of the metabolic disorders of chronic kidney disease (CKD) improve after kidney transplantation, although bone metabolism might remain compromised, which is evidenced by high rates of bone loss, fractures and vascular calcification. Osteocytic bone protein expression is altered in CKD, and this seems to contribute negatively to bone health in these patients. It has been described that FGF-23 and sclerostin expression is increased in children after solid organ transplantation. However, little is known about bone-related proteins expression in adult recipients, which were analyzed prospectively in this study. Transiliac bone biopsies were obtained from 31 adult patients one week before and one year after transplantation. Bone fragments were used for histomorphometric analysis, as well as for bone proteins expression, measured by immunohistochemistry (IH) and multiplex. At baseline, we observed a significant correlation between IH expression and multiplex concentrations for sclerostin and FGF-23. After a successful transplant, there was a decrease in PTH, TRAP5b, bone alkaline phosphatase, FGF-23, OPG and sclerostin. Although serum sclerostin decreased after the transplant, bone content of this protein increased through immunohistochemistry, multiplex and gene expression. We also observed an increase in the bone content and bone expression of phosphorylated beta-catenin, confirming the Wnt pathway inhibition, which was accompanied by RANKL increase and OPG decrease in the bone. A significant decrease in FGF-23 bone concentration was also seen, compatible with the serum decrease. Kidney function recovery after transplant is accompanied by significant changes in many bone proteins expression. Contradictory to the decrease in levels of serum sclerostin, its bone expression, actually, has increased, accompanied by the change of other proteins that confirm the Wnt pathway inhbition. This findings could help to unveil the patophysiology of post transplant bone disease and help to guide the search to new therapies

O tecido ósseo é extremamente complexo que, juntamente com a cartilagem, constitui o sistema esquelético. Tanto os ossos quanto a cartilagem são compostos por tecido metabolicamente ativo com duas funções básicas para o organismo, uma mecânica e outra bioquímica. O impacto do déficit calórico e da perda de peso pode reduzir a massa óssea e mudar a composição corpórea. Na pancreatite crônica alcoólica o paciente relata ingestão alcoólica por longo período, além da referência do alto consumo de cigarros e de uma alimentação deficiente. Os objetivos do presente estudo foram avaliar a frequência da doença osteometabólica, os hábitos alimentares, a frequência de deficiência de vitamina D assim como, se os achados de massa corpórea por densitometria de corpo total se relacionam à deficiência de massa óssea, em indivíduos portadores de pancreatite crônica de etiologia alcoólica. Foram avaliados três grupos de pacientes do sexo masculino com pancreatite crônica alcoólica. Foram divididos de acordo com o resultado da densitometria óssea: 5 pacientes no grupo da osteoporose, 26 no grupo da osteopenia e 8 no grupo normal. Todos os pacientes foram submetidos ao registro alimentar de três dias, mensuração de peso, altura, cintura e quadril, Índice de Massa Corpórea (IMC) e exames laboratoriais. A composição corpórea foi avaliada pela densitometria óssea por raios X de dupla energia (DXA) e por bioimpedância elétrica. 79% dos pacientes do sexo masculino com pancreatite crônica alcoólica tiveram densidade mineral óssea comprometida. Os pacientes que tinham vitamina D prescrita foram excluídos porém nos nossos resultados a maioria dos pacientes apresentavam níveis normais da vitamina. Em relação ao tabagismos, dos pacientes fumavam. Os pacientes com maior comprometimento ósseo eram mais magros,contudo, não houve diferença entre os pacientes de acordo com o IMC. Os pacientes classificados pelo DXA como normais eram mais jovens do que os pacientes com osteopenia e osteoporose. Em síntese, a osteoporose e osteopenia são fontes subvalorizadas de morbidade em pacientes com pancreatites crônicas sendo necessárias diretrizes de gestão de saúde óssea neste grupo de pacientes
The bone tissue is extremely complex, along with cartilage constitutes the skeletal system. Both bones as cartilage are composed of metabolically active tissue with two basic functions for the body, mechanical and biochemistry. The impact of the caloric deficit and weight loss can reduce bone mass and change body composition. In chronic alcoholic pancreatitis patients alcohol intake over a long period, in addition to reference the high consumption of cigarettes and poor nutrition. The objectives were to evaluate the frequency of osteometabolic disease, eating habits, the frequency of vitamin D deficiency and how the body mass found by total body densitometry relate to bone deficiency in individuals with chronic pancreatitis of alcoholic etiology . We evaluated three groups of male patients with chronic pancreatitis alcoholic. They were according to the results of bone densitometry. 5 in osteoporosis group, 26 in the osteopenia group and 8 in the normal group. All patients underwent three-day food record, measurements of weight, height, waist and hip, body mass index (BMI) and laboratory tests. The body composition was evaluated by densitometry by dual energy X-ray absorptiometry (DXA) and electrical bioimpedance. 79% of male patients with alcoholic chronic pancreatitis had compromised bone mineral density. Patients were prescribed vitamin D were excluded however results in the majority of patients had normal levels of the vitamin. Half of all patients smoking. Patients with higher bone involvement were thinner, there was no difference between patients according to BMI. Patients classified as normal by DXA were younger than patients with osteopenia and osteoporosis. In summary, osteoporosis and osteopenia are undervalued sources of morbidity in patients with chronic pancreatitis and necessary health management guidelines bone in this group of patients

Foi comparada a massa óssea e a composição corporal dos hemicorpos dominante e não dominante de um grupo (A) de 16 pessoas com paralisia cerebral com hemiplegia espástica, e de um grupo (B) de 29 voluntários normais por meio da mensuração da massa corporal, estatura e densitometria óssea do corpo total com composição corporal. Foi encontrada diferença estatística significante no conteúdo mineral ósseo dos membros superiores do grupo A e dos membros superiores e inferiores, tronco e total do grupo B; na massa magra dos membros inferiores do grupo A e dos membros superiores e inferiores, tronco e total do grupo B; na massa adiposa dos membros inferiores do grupo A e dos membros superiores e inferiores, tronco e total do grupo B; e no conteúdo mineral ósseo dos membros superiores e inferiores, e total e na massa magra dos membros superiores e inferiores entre os hemicorpos não dominantes dos grupos A e B. Foi encontrada também correlação estatística significante entre o conteúdo mineral ósseo e a massa magra nos grupos A e B em todos os sítios, exceto no tronco dominante do grupo A; e o conteúdo mineral ósseo e a massa corpórea no tronco dominante do grupo A e no membro inferior dominante e bilateral do grupo B
Were compared the bone mass and body composition of the dominant and nondominant hemi bodies in a group (A) of a 16 spastic hemiplegic cerebral palsy and in a group (B) of 27 normal volunteers by weight, height, and densitometry of total body with body composition measure. Were observed significant statistical difference in the bone content mineral of the upper limbs of the group A and of the upper and lower limbs, trunk and total of the group B; in the lean mass of the lower limbs of the group A and of the upper and lower limbs, trunk and total of the group B; in the mass fat of the lower limbs of the group A and of the upper and lower limbs, trunk and total of the group B; and in the bone mineral content of the upper and lower limbs, and total, and in the lean mass of the upper and lower limbs between the hemi bodies no dominant of the A and B groups. Were observed too statistical correlation between the bone mineral content and lean mass in the groups A and B in all locals, except in the trunk of group A; and between the bone mineral content and body mass in the trunk of group A and lower limb dominant and bilateral of group B

UVOD: Artroza šaka predstavlja jednu od najčešćih mišićno-skeletnih bolesti. Manifestuje se bolom, nekada otokom, deformacijom i gubitkom funkcije šaka. Postoje različita mišljenja o povezanosti osteoartroze (OA) i osteoporoze (OP) kao dva najčeša skeletna poremećaja. CILJ: istraživanja je da se utvrde faktori rizika za nastanak OA šaka, uporedi mineralna koštana gustina kod pacijenata sa OA šaka sa kontrolnom grupom i utvrdi značaj metaboličkog sindroma kod pacijenata sa OA šaka. MATERIJAL I METODE: Istraživanje je obavljeno u periodu od jedne godine kod bolesnika sa OA šaka – eksperimentalna grupa, i u kontrolnoj grupi bez OA. OA šaka je definisana na osnovu bola, klinički prisutnih deformiteta šaka kod žena u postmenopauzi starosne dobi od 60-70 godina i radiografskih promena ( drugog do četvrtog stepena prema Kellgren-Lowrencovoj skali). Analizirani su faktori rizika odgovorni za nastanak OA šaka, povezanost OA šaka sa snagom stiska šake, mineralnom koštanom gustinom i metaboličkim sindromom. Analazirirana je i funkcija šake pomoću tri validirana upitnika: Michigan Hand Outcomes Questionnaire (MHQ, Duruoz Hand Indeks (DHI), Health Assessment Questionnaire (PROMIS HAQ). Statistička obrada podataka rađena je u programu SPSS verzija 25. REZULTATI: Prosečna starost pacijentkinja je bila 65,89±3,67 godina. Eksperimentalna i kontrola grupa se statistički razlikuju prema porodičnoj anamnezi o strukturnim promenama zglobova šaka, prema bolnosti šaka u miru, bolnosti šaka pri palpaciji, uzdržavanju od pokreta prstiju šaka, snage stiska šake, metaboličkom sindromu( p<0,001). Kao značajni prediktori za nastanak osteoartroze šaka su se izdvojili pozitivna porodična anamneza o strukturnim promenama za zglobovima šaka i metabolički sindrom ( p<0,001). Utvrđen je veći broj ispitanica sa normalnom koštanom gustinom u kontrolnoj grupi. Eksperimentalna grupa bolesnica imala je lošiju funkciju šake, odnosno lošiji skor primenom validiranih upitnika ( p <0,001). ZAKLJUČAK: Pacijentkinje sa izraženom osteoartrozom šaka imaju smanjenu funkciju šake, češći metabolički sindrom u odnosu na kontrolnu grupu, ali ne i značajno nižu koštanu gustinu.
INTRODUCTION: Arthritis of the hand is one of the most common musculoskeletal disorders. It manifests as pain, sometimes accompanied by swelling and deformities, which may lead to the loss of hand function. However, there is no consensus on the relationship between osteoarthritis (OA) and osteoporosis (OP) as the two most common skeletal disorders. AIMS: The study aim was to determine the risk factors related to the development of OA in the hand, as well as compare the bone density in patients with hand OA (HOA) with that measured in the control group and establish the significance of metabolic syndrome in the HOA group. MATERIAL AND METHODS: The study was conducted over a 12-month period and included a sample comprising of the experimental (patients affected by HOA) and the control (individuals with no evidence of HOA) group. HOA was diagnosed based on the reported pain, clinical evidence of hand deformities in postmenopausal women aged 60−70, and radiological evidence of physiological changes (Grade II to IV, based on the Kellgren-Lowrence scale). The risk factors for the development of HOA were analyzed, along with the link between HOA and hand grip strength, bone mineral density and metabolic syndrome. Analyses also included had function, as determined by three validated questionnaires: Michigan Hand Outcomes Questionnaire (MHQ), Duruoz Hand Index (DHI), and Health Assessment Questionnaire (PROMIS HAQ). Statistical analyses were performed using the SPSS version 25 computer software. RESULTS: The average age of the sample was 65.89±3.67 years. There were statically significant differences between the experimental and the control group with respect to the family history of structural changes in the hand joints, perceived hand pain at rest and when palpated, reluctance to utilize fingers, hand grip strength, and metabolic syndrome (p < 0.001). Family history of structural changes to the hand joints and metabolic syndrome emerged as the strongest predictors of the osteoarthritis of the hand development (p < 0.001). A greater number of the control group members had normal bone mineral density, while the patients assigned to the experimental group had inferior hand function, as determined by the score on the aforementioned validated questionnaires (p < 0.001). CONCLUSION: Postmenopausal women with pronounced osteoarthritis of the hand have reduced hand function, and are more likely to suffer from a metabolic syndrome relative to the control group, while the differences in bone mineral density are not statistically significant.

Osteoporosis is a significant public health problem in the U.S. It not only affects the physical well-being of the older women but also creates a substantial financial burden for the health care system. The mainstay of osteoporosis medications is bisphosphonate treatment of which alendronate and risedronate are the most commonly prescribed in clinical practice. However, there have been no head-to-head randomized controlled trials (RCTs) evaluating the effects of these two bisphosphonates on fracture outcomes. In the absence of RCTs, observational studies are necessary to provide alternative evidence on the comparative effectiveness between alendronate and risedronate on fracture outcomes. However, existing observational studies have provided inconclusive results partially due to residual confounding from unobserved variables such as patients’ health status or behavior. IV analysis may be one method to address unmeasured confounding bias in observational studies. While it has not been applied in bisphosphonate research, it has been used in research on a variety of other prescription medications. In this dissertation, we applied the IV approach with an IV, date of generic alendronate availability, to evaluate the comparative effectiveness between alendronate and risedronate using observational data. This dissertation improved current research in several ways. First, we extended the IV approach to research on bisphosphonates. Second, compared with the current observational studies on bisphosphonates, this dissertation may more accurately estimate the relative effects between alendronate and risedronate because IV analysis is not subject to unmeasured confounding bias. Third, the study results extended the current evidence of the comparative effectiveness between the two most commonly prescribed bisphosphonates. Finally, we proposed and provided empirical evidence of a new IV that might be used for future prescription drug research. The finding of this dissertation can be summarized from three aspects. First, we found that the evidence supported the validity of the date of generic availability as an IV in the study of bisphosphonates. Second, applying IV approach to study the comparative effectiveness of alendronate and risedronate, we found that alendronate and risedronate were comparable to reduce the risk of 12-month non-vertebral fractures in older women. Since generic alendronate is availability on the market while generic risedronate is not, promoting the use of alendronate may help reduce the healthcare cost and not sacrifice the clinical effectiveness. Finally, by comparing the proposed IV with a popular IV-physician preference, we found that both the calendar time IV based on the date of generic availability and the physician preference appeared to be valid. It might be practically easier to use the calendar time IV than the physician preference IV.

Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: Crianças e adolescentes com doenças hereditárias de metabolismo (IEM) estão em risco de osteopenia. As medidas preventivas antes de alcançar o pico da massa óssea são críticas.O objetivo deste trabalho foi avaliar as alterações na densidade mineral óssea (DMO) e outros parâmetros de avaliação óssea em crianças e adolescentes com hiperlactacidémia crónica.Métodos: Os pacientes foram selecionados entre os que tiveram níveis de lactato medidos em Consulta de Doenças Metabólicas do Centro de Referência de Doenças Metabólicas Herdadas - CHUC num período de dez anos (2006-2016). Os critérios de inclusão foram níveis plasmáticos de lactato acima de 2,1 mmol / L em três ou mais visitas diferentes e disponibilidade de DXA óssea. Os processos dos doentes foram revistos, incluindo diagnóstico, tipo de dieta e outras terapias, nível de ambulação, parâmetros bioquímicos plasmáticos e urinários do metabolismo ósseo e DMO.Resultados: Houve hiperlactacidémia crónica em 43 pacientes, correspondendo a 8,1% dos doentes com lactacidémia avaliados no período do estudo. A densitometria óssea estava disponível em 33 doentes (18 meninos) com hiperlactacidémia crónica (76,7%). Oito pacientes (24,2%) apresentavam doença mitocondrial, sete (21,2%), doença de armazenamento de glicogénio tipo 1, cinco (15,2%), aminoacidopatia / acidúria orgânica e três (9,1%), outras doenças hereditárias do metabolismo. Dez pacientes (30,3%) tinham suspeita de IEM. As idades médias na primeira avaliação de lactato e no final do período de estudo foram 4,1 ± 4,3 e 12 ± 5,7 anos, respectivamente. A média de lactacidémia foi de 3,2 ± 2,4mmol / L. Quatorze indivíduos (42,4%) apresentaram acidose. A maioria dos pacientes apresentou valores normais de parâmetros plasmáticos, com exceção da vitamina D, que foi baixa em 62,5% dos 24. Dezesseis pacientes (48,0%) apresentaram DMO da coluna lombar pontuação Z ≤ -2. Nenhuma associação significativa foi encontrada entre DMO e lactacidemia ou níveis de pH.Discussão: Este estudo transversal retrospectivo incluiu 33 pacientes com hiperlactacidémia crónica e um IEM geneticamente comprovado ou suspeito. Níveis elevados de lactato, redução do pH, reabsorção óssea e inibição da atividade osteoblástica. DXA é uma ferramenta bem reconhecida para a investigação óssea em pediatria. Não foram encontradas correlações estatisticamente significativas entre a lactacidémia ou pH da DMO. No entanto, quase metade dos pacientes apresentaram DMO scores Z da coluna lombar ≤ -2 DP. Como na população em geral, a deficiência / insuficiência de vitamina D era comum no grupo de estudo, embora muitos pacientes estivessem sob suplementos.A análise dos parâmetros bioquímicos do metabolismo do fosfo-cálcio nos parâmetros plasmáticos e urinários foi inconclusiva.Conclusão:Embora a presente investigação tenha apontado para um efeito deletério de hiperlactacidémia na DMO, não foi possível demonstrar associação estatística. Este foi um passo preliminar para avaliar os efeitos da hiperlactacidémia crónica na saúde dos ossos no campo do IEM pediátrico.
Introduction: Children and adolescents with inborn errors of metabolism (IEM) are at risk for osteopenia. Preventive measures prior to reaching peak bone mass is critical.We aimed to evaluate the changes in bone mass density (BMD) and other parameters of bone assessment in children and adolescents with chronic hyperlactacidaemia.Methods: Patients were selected among those who had lactate levels measured at the Paediatric Outpatient Clinics of the Reference Centre of Inherited Metabolic Diseases- CHUC in a ten-year period (2006-2016). Inclusion criteria were plasma lactate levels above 2,1 mmol/L in three or more different visits and availability of bone DXA. Patients’ files were reviewed, including diagnosis, special diet and other therapies, ambulation, biochemical plasma and urine parameters of bone metabolism and BMD.Results: Chronic hyperlactacidemia was disclosed in 43 patients, corresponding to 8,1% of the patients with lactacidaemia evaluated in the study period. Bone densitometry was available in 33 patients (18 boys) with chronic hyperlactacidaemia (76,7%). Eight patients (24,2%) had mitochondrial disease, seven (21,2%), glycogen storage disease type 1, five (15,2%), aminoacidopathy/organic aciduria and three (9,1%), other inherited metabolic disorders. Ten patients (30,3%) had suspected IEM. Mean ages at first lactate evaluation and at the end of the study period were 4,1±4,3 and 12±5,7 years-old, respectively. Median lactacidaemia was 3,2±2,4mmol/L. Fourteen individuals (42,4%) displayed acidosis. Most the patients presented normal values of plasma parameters, except for vitamin D, which was low in 62,5% of 24. Sixteen patients (48,0%) exhibited lumbar spine BMD Z-Score SD ≤-2. No significant association was found between BMD and lactacidaemia or pH levels. Discussion: This cross-sectional retrospective study included 33 patients with chronic hyperlacticaemia and an IEM genetically proved or suspected. High lactate levels, lowering pH, causes bone reabsorption and inhibits osteoblastic activity. DXA exam is a well-recognized tool for bone investigation in Paediatrics. No statistically significant correlations were found between BMD lactacidaemia or pH. Nevertheless, almost half of the patients presented lumbar spine BMD Z-scores ≤ -2 SD. As in the general population, Vitamin D deficiency/insufficiency was common in the study group, although many patients were under supplements.Analysis of the biochemical phospho-calcium metabolism plasma and urine parameters results was inconclusive.Conclusion: Although the present investigation pointed to a deleterious effect of hyperlactacidae-mia on BMD, a statistical association could not be proved. This was a preliminary step to ad-dress the effects of chronic hyperlactacidaemia on bone heath in the field of paediatric IEM.

背景：纤维化是各种因素导致肾脏慢性损伤的最终病理过程，是决定肾功能转归的关键因素。肌纤维母细胞作为构成肾脏纤维化组织的主要细胞成分，其来源尚不清楚。本研究认为骨髓来源的巨噬细胞向肌纤维母细胞转分化（MMT）可能是肾脏纤维化中肌纤维母细胞的主要来源。我们分别在慢性肾脏病患者的肾活检组织和小鼠单侧输料管梗阻模型（UUO）中验证这一假说。
方法：我们用激光共聚焦技术和流式细胞染色的方法检测小鼠UUO肾脏和患者肾活检组织中的MMT细胞(F4/80⁺α-SMA⁺或CD68⁺α-SMA⁺)。为了验证骨髓来源的MMT在肾纤维化中的重要作用，UUO模型分别在以下小鼠进行：1）去除骨髓的C57BL/6J小鼠，给予或不给予绿色荧光蛋白（GFP）标记的骨髓细胞移植；2）GFP⁺骨髓的嵌合体小鼠；3）巨噬细胞敲除或不敲除的lysM-Cre/DTR小鼠；4）GFP⁺Smad3⁺/⁺ 或GFP⁺Smad3⁻/⁻骨髓的嵌合体小鼠。我们用实时定量PCR和Western blot检测小鼠肾组织collagen-I和α-SMA水平。另外，我们观察MMT细胞和PDGFR-β⁺ pericytes, CD45⁺collagen I⁺ fibrocytes的关系。最后，通过观察GFP⁺Smad3⁻/⁻骨髓嵌合体小鼠UUO模型肾纤维化程度和TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT的不同进一步探索TGF-β/Smad3通路对MMT的影响。
结果：去除骨髓后，肾脏collagen-I沉积和α-SMA⁺肌纤维母细胞生成显著受抑制，骨髓细胞移植可以恢复肾脏纤维化，免疫荧光染色显示嵌合体小鼠中多数（80-90%）肌纤维母细胞来自于骨髓巨噬细胞转分化。同时，在白喉霉素诱导的巨噬细胞敲除小鼠中，50-60%巨噬细胞被去除，伴有纤维化明显减少，并且和MMT细胞显著减少相关。进一步验证巨噬细胞通过MMT直接参与肾脏纤维化过程。患者肾活检组织亦可见不同数目MMT细胞，纤维化活跃组织中MMT细胞可占到肌纤维母细胞总数的80%。另外，我们发现无论在小鼠模型还是患者肾活检组织中，多数MMT细胞表达pericyte（PDGFR-β⁺）和fibrocyte（CD45⁺collagen-I⁺）标记物。Smad3⁻/⁻骨髓嵌合体小鼠肾纤维化程度明显低于Smad3⁺/⁺骨髓嵌合体组，TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT明显低于不敲除组，提示TGF-β/Smad3通路在MMT过程中起重要作用。
结论：骨髓来源的MMT是肾纤维化组织中肌纤维母细胞的主要来源，TGF-β/Smad3 通路在MMT 过程中起重要作用。
Background: Fibrosis is the ultimate pathological feature and determinant process for chronic kidney disease (CKD) regardless of the underlying etiology. Myofibroblasts are a key cell type in renal fibrosis by producing excessive collagen matrix. However, the origin of myofibroblasts during renal fibrosis remains largely controversial. This thesis tested the hypothesis that bone marrow (BM)-derived macrophage myofibroblast transition (MMT) may be a key pathway leading to renal fibrosis in patients with CKD and in a mouse model of unilateral ureteral obstructive nephropathy (UUO).
Methods: Renal fibrosis was assessed by expression of fibrotic marker collagen I and α-SMA using real-time PCR and western-blot analysis. MMT was determined in both mouse and human kidneys by confocal microscopy and flow cytometry with α-SMA⁺F4/80⁺ (or CD68⁺). The critical role of BM-derived MMT in renal fibrosis was examined in a mouse model of UUO, with various conditions: 1) BM depletion followed by BM transplantation (BMT) with GFP⁺ BM cells; 2) in GFP⁺ BM chimeric mice; 3) in lysM-Cre/DTR mice with or without inducible macrophage deletion; 4) in GFP⁺Smad3⁺/⁺ or GFP⁺Smad3⁻/⁻ BM chimeric mice. In addition, MMT was also validated in renal biopsy tissues from patients with different forms of CKD. Further more, we also studied the relationship between MMT and PDGFR-β⁺ pericytes or CD45⁺collagen I⁺ fibrocytes in both human and mouse fibrotic kidneys. Finally, mechanisms of MMT was examined in the UUO kidney induced in GFP⁺Smad3⁻/⁻ BM chimeric mice and in BM macrophages lacking TGF-β receptor II or Smad3.
Results: As described in Chapter III, mice with BM deletion were protected from renal fibrosis as demonstrated by blocking α-SMA⁺ myofibroblasts and collagen I accumulation. In contrast, BMT restored renal fibrosis in UUO kidney, demonstrating the critical role for BM cells in renal fibrosis. Importantly, the majority (85-90%) of α-SMA⁺ myofibroblasts were derived from BM macrophages as identified by GFP⁺F4/80⁺α-SMA⁺ revealing BM-macrophages given rise to myofibroblasts via MMT during kidney fibrosis. Similarly, MMT appeared as a major pathway of myofibroblast origin in patients with CKD, accounting for up to 80% of total myofibroblasts in the active stage of tissue fibrosis and fibrocellular crescents. To test the function role of macrophages in renal fibrosis via MMT, macrophages were conditionally deleted from the UUO kidneys in lysM-Cre/DTR mice as shown in Chapter IV, deletion (50-60%) of macrophages resulted in inhibition of MMT and renal fibrosis. Unexpectedly, most MMT cells (80-90%) were shown to co-express the pericyte marker (PDGFR-β⁺) and fibrocyte markers (CD45⁺collagen I⁺) in both human CKD and UUO (Chapter V), suggesting a BM macrophage origin for pericytes and fibrocytes during renal fibrosis. Finally, TGF-β/Smad3 appeared to be a mechanism driven MMT because mice and BM macrophages lacking either Smad3 or TβRII were protected against MMT and progressive renal fibrosis in the UUO kidney and in vitro.
Conclusions: MMT is derived from BM macrophages and regulated by TGF-β/Smad3. MMT is a major pathway of myofibroblast origin during renal fibrosis in both human and animal model of CKD.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Wang, Shuang.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 161-179).
Abstracts also in Chinese.
Chapter ABSTRACT --- p.ii
Chapter DECLARATION --- p.viii
Chapter ACKNOWLEDGEMENTS --- p.ix
Chapter TABLE OF CONTENTS --- p.xi
Chapter LIST OF ABBREVIATION --- p.xv
Chapter LIST OF FIGURES AND TABLES --- p.xvii
Chapter CHAPTER I --- p.1
INTRODUCTION --- p.1
Chapter 1. 1 --- Renal fibrosis and myofibroblasts --- p.2
Chapter 1. 1. 1 --- Pathology of renal fibrosis --- p.2
Chapter 1. 1. 2 --- The generation and modulation of myofibroblasts. --- p.3
Chapter 1. 1. 2. 1 --- EMT and EndMT --- p.5
Chapter 1. 1. 2. 2 --- Pericytes --- p.8
Chapter 1. 1. 2. 3 --- Fibrocytes --- p.16
Chapter 1. 2 --- Role of macrophage in fibrogenesis --- p.21
Chapter 1. 3 --- TGF-β signaling pathway in renal fibrosis --- p.23
Chapter 1. 3. 1 --- TGF-β superfamily --- p.23
Chapter 1. 3. 2 --- TGF-β/Smad signaling pathway --- p.24
Chapter CHAPTER II --- p.29
MATERIALS AND METHODS --- p.29
Chapter 2. 1 --- Materials --- p.30
Chapter 2. 1. 1 --- Regents and equipments --- p.30
Chapter 2. 1. 1. 1 --- Regents and equipment for mouse genotyping --- p.30
Chapter 2. 1. 1. 2 --- Regents and equipments for real-time PCR --- p.30
Chapter 2. 1. 1. 3 --- Reagents and equipments for immunohistochemistry staining --- p.31
Chapter 2. 1. 1. 4 --- Reagents and equipment for flow cytometry --- p.32
Chapter 2. 1. 2 --- Buffer --- p.32
Chapter 2. 1. 2. 1 --- Buffers for immunohistochemistry and immunofluorescence staining --- p.32
Chapter 2. 1. 2. 2 --- Buffers for western blot --- p.35
Chapter 2. 1. 3 --- Sequences of primers for genotyping and real-time PCR --- p.41
Chapter 2. 1. 4 --- Antibodies --- p.42
Chapter 2. 2 --- Methods --- p.44
Chapter 2. 2. 1 --- Generation of gene modified mice --- p.44
Chapter 2. 2. 2 --- Bone marrow transplantation --- p.45
Chapter 2. 2. 3 --- Conditional macrophage deletion --- p.45
Chapter 2. 2. 4 --- Unilateral ureteral obstruction (UUO) mouse model --- p.46
Chapter 2. 2. 5 --- Histology and immunohistochemistry --- p.46
Chapter 2. 2. 5. 1 --- Processing paraffin sections --- p.46
Chapter 2. 2. 5. 2 --- Deparaffinization and hydration --- p.47
Chapter 2. 2. 5. 3 --- Blocking endogenous peroxidase --- p.47
Chapter 2. 2. 5. 4 --- Antigen retrieval --- p.48
Chapter 2. 2. 5. 5 --- Antigen and antibody reaction --- p.48
Chapter 2. 2. 5. 6 --- Detection of target signals --- p.49
Chapter 2. 2. 5. 7 --- Quantification of immunohistochemistry staining --- p.49
Chapter 2. 2. 6 --- Immunofluorescence staining and confocal microscopy analysis --- p.49
Chapter 2. 2. 6. 1 --- Processing tissue for immune-fluorescent (IF) staining --- p.49
Chapter 2. 2. 6. 2 --- Serum blocking --- p.50
Chapter 2. 2. 6. 3 --- Antigen antibody reaction --- p.50
Chapter 2. 2. 6. 4 --- Signal detection --- p.51
Chapter 2. 2. 7 --- Flow cytometry --- p.52
Chapter 2. 2. 7. 1 --- Preparation of single cell suspension --- p.52
Chapter 2. 2. 7. 2 --- Cell fixation and permeabilization --- p.53
Chapter 2. 2. 7. 3 --- Staining --- p.53
Chapter 2. 2. 7. 4 --- Signal detection and analysis --- p.54
Chapter 2. 2 .8 --- Real time PCR --- p.55
Chapter 2. 2. 8. 1 --- Total RNA extraction --- p.55
Chapter 2. 2. 8. 2 --- Reverse transcription --- p.56
Chapter 2. 2. 8. 3 --- Real-time PCR --- p.57
Chapter 2. 2. 8. 4 --- Analysis of real-time PCR --- p.57
Chapter 2. 2. 9 --- Western blot --- p.58
Chapter 2. 2. 9. 1 --- Protein extraction from tissue --- p.58
Chapter 2. 2. 9. 2 --- Protein concentration measurement --- p.59
Chapter 2. 2. 9. 3 --- SDS-PAGE electrophoresis --- p.59
Chapter 2. 2. 9. 4 --- Protein transfer --- p.60
Chapter 2. 2. 9. 5 --- Blocking --- p.61
Chapter 2. 2. 9. 6 --- Antibodies incubation and signal detection --- p.62
Chapter 2. 2. 9. 7 --- Stripping --- p.62
Chapter CHAPTER III --- p.63
EVIDENCE FOR MMT AS A NEW PATHWAY OF MYOFIBROBLAST ORIGIN IN RENAL FIBROSIS --- p.63
Chapter 3. 1 --- Introduction --- p.64
Chapter 3. 2 --- Materials and methods --- p.65
Chapter 3. 2. 1 --- Human renal biopsy tissues --- p.65
Chapter 3. 2. 2 --- Experimental design --- p.65
Chapter 3. 2. 3 --- Bone marrow transplantation and GFP⁺ BM chimeric mice --- p.66
Chapter 3. 2. 4 --- Immunohistochemistry --- p.66
Chapter 3. 2. 5 --- Immunofluorescence and confocal microscopy analysis --- p.67
Chapter 3. 2. 6 --- Real-time PCR --- p.68
Chapter 3. 2. 7 --- Western blot analysis --- p.68
Chapter 3. 2. 8 --- Flow cytometry --- p.68
Chapter 3. 3 --- Results --- p.69
Chapter 3. 3. 1 --- BM-derived myofibroblasts play a key role in renal fibrosis in a mouse model of UUO --- p.69
Chapter 3. 3. 1. 1 --- α-SMA⁺ myofibroblasts are derived from BM and determine renal fibrosis in a mouse model of UUO --- p.69
Chapter 3. 3. 1. 2 --- BM as a major source of collagen production in a mouse model of UUO --- p.73
Chapter 3. 3. --- 2 Evidence for BM derived macrophage-myofibrobalst transition (MMT) in a mouse model of UUO --- p.77
Chapter 3. 3. 2. 1 --- Characterization of GFP⁺ BM chimeric mice --- p.77
Chapter 3. 3. 2. 2 --- Evidence for bone marrow-derived MMT is the major source of myofibroblast origin in the UUO kidney --- p.79
Chapter 3. 3. 3 --- Evidence for MMT in human fibrotic kidney tissues --- p.84
Chapter 3. 3. 4 --- M2 macrophage is the predomimant phenotype of macrophages in the fibrotic kidney of UUO mouse model. --- p.88
Chapter 3. 4 --- Discussion --- p.90
Chapter 3. 5 --- Conclusion --- p.93
Chapter CHAPTER IV --- p.94
Chapter GE --- CONDITIONAL MACROPHA DELETION INHIBITS MMT AND RENAL FIBROSIS --- p.94
Chapter 4. 1 --- Introduction --- p.95
Chapter 4. 2 --- Materials and methods --- p.98
Chapter 4. 2. 1 --- Generation of lysM-Cre/DTR mice --- p.98
Chapter 4. 2. 2 --- Conditional deletion of macrophage --- p.98
Chapter 4. 2. 3 --- Unilateral Ureteral Obstruction (UUO) mouse model --- p.98
Chapter 4. 2. 4 --- Real-time PCR --- p.99
Chapter 4. 2. 5 --- Western blot analysis --- p.99
Chapter 4. 2. 6 --- Immunohistochemisty --- p.99
Chapter 4. 2. 7 --- Immunofluorescence --- p.99
Chapter 4. 3 --- Results --- p.100
Chapter 4. 3. 1 --- Characterization of lysM-Cre/DTR mice --- p.100
Chapter 4. 3. 2 --- Conditional deletion of macrophage in a mouse model of UUO --- p.101
Chapter 4. 3. 3 --- Conditional deletion of macrophage suppresses α-SMA⁺ myofibroblast accumulation in a mouse model of UUO --- p.104
Chapter 4. 3. 4 --- Conditional deletion of macrophage inhibits collagen I production in a mouse model of UUO --- p.106
Chapter 4. 3. 5 --- Conditional deletion of macrophage inhibits renal fibrosis through reducing MMT cells in a mouse model of UUO --- p.108
Chapter 4. 4 --- Discussion --- p.111
Chapter 4. 5 --- Conclusion --- p.113
Chapter CHAPTER V --- p.114
MMT CELLS SHARE PERICYTE AND FIBROCYTE PHENOTYPES --- p.114
Chapter 5. 1 --- Introduciton --- p.115
Chapter 5. 2 --- Materials and methods --- p.116
Chapter 5. 2. 1 --- Human renal biopsy tissues --- p.116
Chapter 5. 2. 2 --- Animals and UUO mouse model --- p.116
Chapter 5. 2. 3 --- Immunofluorescence (IF) --- p.116
Chapter 5. 2. 4 --- Flow cytometry --- p.117
Chapter 5. 3 --- Results --- p.119
Chapter 5. 3. 1 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney of UUO model --- p.119
Chapter 5. 3. 2 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.124
Chapter 5. 3. 3 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney of UUO model --- p.126
Chapter 5. 3. 4 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.129
Chapter 5. 4 --- Dscussion --- p.131
Chapter 5. 5 --- Conclusion --- p.133
Chapter CHAPTER VI --- p.134
SMAD3 MEDIATES MMT DURING RENAL FIBROSIS --- p.134
Chapter 6. 1 --- Introduction --- p.135
Chapter 6. 2 --- Materials and methods --- p.137
Chapter 6. 2. 1 --- Generation of Smad3⁺/⁺ and Smad3⁻/⁻ BM-Chimeric mice --- p.137
Chapter 6. 2. 2 --- Generation of TbRII disrupted BM macrophages and Smad3⁻/⁻ BM macrophages --- p.137
Chapter 6. 2. 3 --- UUO mouse model --- p.138
Chapter 6. 2. 4 --- Cell culture --- p.138
Chapter 6. 2. 5 --- Real-time PCR --- p.139
Chapter 6. 2. 6 --- Western blot analysis --- p.139
Chapter 6. 2. 7 --- Immunohistochemistry (IHC) --- p.139
Chapter 6. 2. 8 --- Immunofluorescence (IF) --- p.139
Chapter 6. 2. 9 --- Flow cytometry --- p.140
Chapter 6. 3 --- Result --- p.141
Chapter 6. 3. 1 --- Genotyping of Smad3 WT and Smad3 KO mice --- p.141
Chapter 6. 3. 2 --- Smad3 knockout inhibits TGF-β1 induced MMT in vitro --- p.142
Chapter 6. 3. 3 --- Disruption of TbRII inhibits TGF-β1 induced MMT in vitro --- p.145
Chapter 6. 3. 4 --- Deletion of BM Smad3 inhibits α-SMA expression in the UUO kidney --- p.147
Chapter 6. 3. 5 --- Deletion of BM Smad3 inhibits collagen-I production in the UUO kidney --- p.149
Chapter 6. 3. 6 --- Inhibition of MMT is a mechanism by which BM Smad3 deficiency inhibits renal fibrosis in a mouse model of UUO --- p.150
Chapter 6. 4 --- Discussion --- p.153
Chapter 6. 5 --- Conclusion --- p.154
Chapter CHAPTER VII --- p.155
SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.155
Chapter 7. 1 --- Summary and discussion --- p.157
Chapter 7. 1. 1 --- MMT is a major pathway of myofibroblast origin in renal fibrosis --- p.157
Chapter 7. 1. 2 --- MMT cells shares both pericyte and fibrocyte phenotypes in renal fibrosis --- p.157
Chapter 7. 1. 3 --- TGF-β/Smad3 is a key mechanism of MMT in renal fibrosis --- p.158
Chapter 7. 2 --- Conclusion --- p.160
Chapter REFERENCES --- p.161

Hung Wing Yin Vivian.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 128-142).
Abstracts in English and Chinese ; appendix in Chinese.
ABSTRACT --- p.i
ABSTRACT (in Chinese) --- p.iv
ACKNOWLEDGMENT --- p.vii
TABLE OF CONTENTS --- p.viii
LIST OF TABLES --- p.xiv
LIST OF FIGURES --- p.xvi
LIST OF ABBREVIATIONS --- p.xix
Chapter I. --- INTRODUCTION --- p.1
Chapter 1.1. --- Scoliosis --- p.1
Chapter 1.1.1. --- Classification of scoliosis --- p.1
Chapter 1.1.2. --- Idiopathic scoliosis --- p.1
Chapter 1.1.3. --- Clinical examination --- p.2
Chapter 1.1.4. --- Curve pattern --- p.2
Chapter 1.2. --- Etiology of AIS --- p.3
Chapter 1.2.1. --- Prevalence of AIS --- p.5
Chapter 1.2.2. --- Anthropometric Measurement in AIS --- p.5
Chapter 1.2.3. --- Bone mass --- p.6
Chapter 1.2.4. --- Bone mineral density measurements --- p.6
Chapter 1.2.5. --- Osteopenia in AIS --- p.7
Chapter 1.3. --- Natural history ofAIS --- p.8
Chapter 1.3.1. --- Curve progression --- p.9
Chapter 1.3.2. --- Treatment of scoliosis --- p.11
Chapter 1.4. --- Research questions --- p.12
Chapter 1.5. --- Objectives --- p.13
Chapter II. --- METHODOLOGY --- p.20
Chapter 2.1 --- Study Design --- p.20
Chapter 2.2 --- Subject recruitment --- p.20
Chapter 2.2.1 --- AIS patients --- p.20
Chapter 2.2.2 --- Inclusion criteria --- p.20
Chapter 2.2.3 --- Exclusion criteria --- p.20
Chapter 2.2.4 --- Informed consent --- p.21
Chapter 2.3 --- Grouping for chronological age --- p.21
Chapter 2.4 --- Radiography assessments --- p.21
Chapter 2.4.1 --- Cobb angle measurement --- p.21
Chapter 2.4.2 --- Curve pattern --- p.22
Chapter 2.4.3 --- Risser grade --- p.22
Chapter 2.5 --- Definition of curve progression --- p.22
Chapter 2.6 --- Bone mineral density (BMD) measurements --- p.23
Chapter 2.6.1 --- Dual energy X-ray Absorptiometry (DXA) --- p.23
Chapter 2.6.2 --- Peripheral quantitative computed tomography (pQCT) --- p.24
Chapter 2.6.3 --- Definition of osteopenia or low bone mass --- p.24
Chapter 2.7 --- Anthropometric measurements --- p.25
Chapter 2.7.1 --- Body height --- p.25
Chapter 2.7.2 --- Body weight --- p.26
Chapter 2.7.3 --- Arm span --- p.26
Chapter 2.7.4 --- Sitting height --- p.27
Chapter 2.8 --- Family history --- p.27
Chapter 2.9 --- Menstrual status --- p.27
Chapter 2.10 --- Medication and fracture history --- p.27
Chapter 2.11 --- Statistical analysis --- p.27
Chapter 2.11.1 --- Sample size power calculation --- p.28
Chapter 2.11.2 --- Student t test --- p.28
Chapter 2.11.3 --- Paired t-test --- p.28
Chapter 2.11.4 --- Predicting the incidence of curve progression --- p.28
Chapter 2.11.4.1 --- Predictive outcome --- p.28
Chapter 2.11.4.2 --- Potential risk factors --- p.28
Chapter 2.11.4.3 --- Coding system for categorical variables --- p.29
Chapter 2.11.4.4 --- Univariate analysis --- p.30
Chapter 2.11.4.5 --- Logistic regression --- p.30
Chapter 2.11.4.6 --- Receiver operating characteristics (ROC) curves --- p.32
Chapter III. --- RESULTS --- p.54
Chapter 3.1 --- Patients Characteristics --- p.54
Chapter 3.1.1 --- Sample size --- p.54
Chapter 3.1.2 --- Distribution of patient characteristics --- p.54
Chapter 3.1.3 --- Drop out --- p.54
Chapter 3.1.4 --- Prevalence of osteopenia (BMDage-adjusted ≤ -1) and low bone mass (BMCage-adjusted ≤ -1) --- p.55
Chapter 3.1.5 --- Comparison between the BMD of the bilateral hip and tibia --- p.55
Chapter 3.2 --- Comparison of AIS patients with osteopenia and with normal bone status --- p.55
Chapter 3.3 --- Univariate analysis --- p.56
Chapter 3.3.1 --- Growth related factors --- p.56
Chapter 3.3.2 --- "Skeletal related parameters (areal BMD, volumetric BMD and BMC)" --- p.56
Chapter 3.3.2.1 --- DXA lumbar spine --- p.56
Chapter 3.3.2.2 --- DXA proximal femur at the convex-side hip --- p.56
Chapter 3.3.2.3 --- DXA proximal femur at the concave-side hip --- p.57
Chapter 3.3.2.4 --- pQCT at non-dominant distal radius --- p.57
Chapter 3.3.2.5 --- pQCT - vBMD at convex-side distal tibia --- p.57
Chapter 3.3.2.6 --- pQCT - vBMD at concave-side distal tibia --- p.58
Chapter 3.3.3 --- Curve related factors --- p.58
Chapter 3.3.4 --- Anthropometrics parameters --- p.58
Chapter 3.3.5 --- Family history --- p.58
Chapter 3.3.6 --- Summary of univariate analysis --- p.59
Chapter 3.4 --- Logistic regression model (single factor) --- p.59
Chapter 3.5 --- Logistic regression model (multiple factors) --- p.60
Chapter 3.5.1 --- BMD inclusive model --- p.60
Chapter 3.5.2 --- BMC inclusive model --- p.61
Chapter 3.5.3 --- Conventional model --- p.63
Chapter 3.6 --- ROC curve --- p.63
Chapter 3.6.1 --- BMD inclusive model --- p.64
Chapter 3.6.2 --- Conventional model --- p.64
Chapter 3.7 --- Predictive equation obtained from different logistic regression models --- p.64
Chapter 3.7.1 --- BMD inclusive model --- p.65
Chapter 3.7.2 --- Conventional model --- p.65
Chapter IV. --- DISCUSSION --- p.105
Chapter 4.1 --- Prognostic factors for curve progression --- p.105
Chapter 4.1.1 --- Well-known prognostic factors --- p.105
Chapter 4.1.1.1 --- Growth-related factors --- p.106
Chapter 4.1.1.2 --- Initial curve magnitude --- p.107
Chapter 4.1.2 --- A new predictor 一 Osteopenia --- p.107
Chapter 4.2 --- Non-significant prognostic factors for curve progression --- p.109
Chapter 4.2.1 --- Anthropometric parameters --- p.109
Chapter 4.2.2 --- Family History --- p.110
Chapter 4.2.3 --- Curve pattern --- p.110
Chapter 4.3 --- Predictive model --- p.111
Chapter 4.4 --- Comparison of predictive models between BMD inclusive model and conventional model derived from our population --- p.115
Chapter 4.5 --- Possible relationship between osteopenia and etiopathogensis of AIS --- p.116
Chapter 4.6 --- Axial measurement has a better predictive power in curve progression than peripheral measurement --- p.117
Chapter 4.7 --- Discordance of BMD in bilateral hips --- p.118
Chapter 4.8 --- Method justifications --- p.119
Chapter 4.8.1 --- Definition of curve progression --- p.119
Chapter 4.8.2 --- Incidence of progression as the outcome of prediction --- p.119
Chapter 4.8.3 --- Selection on bone densitometers --- p.119
Chapter 4.9 --- Clinical significance --- p.121
Chapter 4.10 --- Limitations and Future Studies --- p.122
Chapter 4.10.1 --- Limited follow-up time --- p.122
Chapter 4.10.2 --- No defined cutoff value for 226}0´ببosteopenia 226}0ح or low BMC in paediatric area --- p.122
Chapter 4.10.3 --- Predictive model could only applied in local population --- p.122
Chapter 4.10.4 --- Intrinsic error in Risser grade measurement --- p.123
Chapter 4.10.5 --- Further studies --- p.123
Chapter 4.10.5.1 --- Validation of the newly developed predictive model --- p.123
Chapter 4.10.5.2 --- Possible intervention of osteopenia --- p.124
Chapter 4.10.5.3 --- Long term follow-up BMD measurements and fracture risk in AIS patients --- p.124
Chapter 4.10.5.4 --- Discordance of bilateral hips BMD contributed by the shift of center of gravity --- p.125
Chapter 4.10.5.5 --- Axial QCT can be an alternative method in assessing BMDin scoliotic patients --- p.125
Chapter V. --- CONCLUSION --- p.126
Chapter VI. --- APPENDIX --- p.127
Chapter VII. --- BIBLIOGRAPHY --- p.128
Chapter VIII. --- CONFERENCE PUBLICATIONS --- p.142

目前，骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展，採用核酸干擾策略進行治療的很多問題已被解決。然而，小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大，其系統給藥後可能對其他非骨組織產生副作用。所以，亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統，以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞，使其具有高度的細胞選擇性。在該研究中，我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。
靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統：首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備（天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑，ζ電位，包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。
實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右，其包封率可高達80%。該遞送系統較穩定，可使攜載的小干擾核酸具有較高的基因沉默效率，而且沒有明顯的細胞毒性。體內試驗表明，該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因，且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞，避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp，Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。
靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統：將針對大鼠成骨細胞（ROS 17/2.8細胞系）進行正向篩選，大鼠肝細胞（BRL-3A細胞系）和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑，ζ電位，包封率和形態學進行考察。在體外評價實驗中，考察了該遞送系統的選擇性，細胞毒性，基因沉默效率以及細胞攝取機制。在體內實驗中，對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。
實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm，其電勢為-23 ± 2 mV，包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系（靶向細胞）中的攝取， 然而在BRL-3A 細胞系（非靶向細胞）中攝入很少。該遞送系統沒有明顯細胞毒性，在10 nM小干擾核酸的低濃度下，體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈，降低其被肝組織的攝取。在肝组织冰凍切片中，肝血竇和肝細胞中沒有明顯的小干擾核酸分佈，進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞，避免被前破骨細胞/破骨細胞吞噬。
重要意義：本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑，實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下，首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具，為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。
Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study.
(AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models.
The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo.
L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo.
The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo.
Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Wu, Heng.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 130-142).
Abstract also in Chinese.
Acknowledgements --- p.i
Abstract --- p.iii
論文摘要 --- p.vi
Table of contents --- p.ix
Publications --- p.xiv
List of tables --- p.xvi
List of figures --- p.xvii
List of abbreviations --- p.xxi
Chapter One Introduction --- p.1
Chapter 1.1 --- Great challenges in skeletal disorders --- p.2
Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3
Chapter 1.2.1 --- Mechanism of RNAi --- p.3
Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4
Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7
Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11
Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12
Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13
Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15
Chapter 1.3.2.1 --- Physiological property --- p.15
Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16
Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18
Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18
Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20
Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20
Chapter 1.4.2.2 --- Polymers --- p.22
Chapter 1.4.2.3 --- Targeted delivery system --- p.23
Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24
Chapter 1.6 --- Objective of present study --- p.25
Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26
Chapter 2.1 --- Introduction --- p.27
Chapter 2.2 --- Materials and Methods --- p.28
Chapter 2.2.1 --- Materials --- p.28
Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29
Chapter 2.2.3 --- Development of formulation --- p.30
Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30
Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30
Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30
Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33
Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33
Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33
Chapter 2.2.5.3 --- Stability in serum --- p.33
Chapter 2.3 --- Results --- p.34
Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34
Chapter 2.3.2 --- Development of formulation --- p.37
Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38
Chapter 2.3.4 --- Stability in serum --- p.38
Chapter 2.4 --- Discussion --- p.40
Chapter 2.5 --- Conclusion --- p.42
Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43
Chapter 3.1 --- Introduction --- p.44
Chapter 3.2 --- Materials and Methods --- p.45
Chapter 3.2.1 --- Materials --- p.45
Chapter 3.2.2 --- Biological evaluation in vitro --- p.46
Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46
Chapter 3.2.2.2 --- Cell culture --- p.46
Chapter 3.2.2.3 --- Cellular uptake --- p.47
Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47
Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48
Chapter 3.2.3 --- Cytotoxicity --- p.49
Chapter 3.2.4 --- Tissue distribution --- p.50
Chapter 3.2.4.1 --- Experimental design --- p.50
Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50
Chapter 3.2.4.3 --- Quantitative Analysis --- p.50
Chapter 3.2.5 --- Localization of siRNA in liver --- p.51
Chapter 3.2.5.1 --- Experimental design --- p.51
Chapter 3.2.5.2 --- Histochemisty analysis --- p.51
Chapter 3.2.6 --- Gene silencing in tissues --- p.52
Chapter 3.2.6.1 --- Experimental design --- p.52
Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52
Chapter 3.2.6.3 --- Western blot analysis --- p.52
Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53
Chapter 3.2.7.1 --- Experimental design --- p.53
Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53
Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54
Chapter 3.2.8.1 --- Experimental design --- p.54
Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54
Chapter 3.2.9 --- Statistical analysis --- p.55
Chapter 3.3 --- Results --- p.56
Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56
Chapter 3.3.2 --- Cellular uptake --- p.57
Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57
Chapter 3.3.4 --- Cytotoxicity --- p.59
Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60
Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62
Chapter 3.3.7 --- Localization of siRNA in liver --- p.63
Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64
Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65
Chapter 3.3.10 --- Gene silencing at cellular level --- p.71
Chapter 3.4 --- Discussion --- p.74
Chapter 3.5 --- Conclusion --- p.77
Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78
Chapter 4.1 --- Introduction --- p.79
Chapter 4.2 --- Materials and Methods --- p.80
Chapter 4.2.1 --- Materials --- p.80
Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80
Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81
Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81
Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82
Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82
Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83
Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83
Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83
Chapter 4.2.3 --- Development of formulation --- p.84
Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84
Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85
Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85
Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86
Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86
Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87
Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87
Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88
Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88
Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88
Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88
Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89
Chapter 4.3 --- Results --- p.90
Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90
Chapter 4.3.2 --- Formulation development --- p.93
Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95
Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96
Chapter 4.4 --- Discussion --- p.98
Chapter 4.5 --- Conclusion --- p.101
Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102
Chapter 5.1 --- Introduction --- p.103
Chapter 5.2 --- Materials and Methods --- p.103
Chapter 5.2.1 --- Materials --- p.103
Chapter 5.2.2 --- Biological evaluation in vitro --- p.104
Chapter 5.2.2.1 --- Cell culture --- p.104
Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105
Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105
Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105
Chapter 5.2.3 --- Cytotoxicity --- p.106
Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106
Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106
Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107
Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107
Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107
Chapter 5.2.5.1 --- Experimental design --- p.107
Chapter 5.2.5.2 --- Tissue distribution --- p.108
Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108
Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108
Chapter 5.2.6 --- Statistical analysis --- p.109
Chapter 5.3 --- Results --- p.109
Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109
Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111
Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112
Chapter 5.3.4 --- Cytotoxicity --- p.113
Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113
Chapter 5.3.6 --- Tissue distribution --- p.118
Chapter 5.3.7 --- Localization of siRNA in liver --- p.119
Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120
Chapter 5.4 --- Discussion --- p.123
Chapter 5.5 --- Conclusion --- p.125
Chapter Chapter Six --- Summary of the study and future research --- p.126
Chapter 6.1 --- Summary of the study --- p.127
Chapter 6.2 --- Future research --- p.128
References --- p.130

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017.
Background Abnormalities of mineral bone disease have been consistently associated with adverse clinical outcomes in patients with chronic kidney disease (CKD). The consequences of these changes have also been shown to differ across races. However, in Africa the impact of derangements of CKD -mineral and bone disorder (CKD-MBD) on patients with CKD is largely unknown. In addition, studies from the USA have reported racial variations in markers of CKD and it remains unclear whether genetic factors may explain this discrepancy in the levels of biochemical markers of CKD-MBD across ethnic groups. Therefore, this study has been conducted to determine the existence of racial differences in the levels of fibroblast growth factor 23(FGF23) and traditional markers of mineral bone metabolism in a heterogeneous African CKD population, and to provide important insights into the pattern and genetic variability of CKD-MBD in sub-Saharan Africa. Methods This was a cross sectional multicenter study carried out from April 2015 to May 2016, involving two hundred and ninety three CKD patients from three renal units in Johannesburg, South Africa. The retrospective arm of this study involved two hundred and thirteen patients undergoing maintenance haemodialysis (MHD) from two dialysis centers in Johannesburg between January 2009 and March 2016. The first part of this study described the pattern of CKD-MBD in MHD patients using traditional markers of CKD-MBD. The second part of the study looked into the spectrum of CKD-MBD and racial variations in markers of CKD-MBD in pre dialysis and dialysis patients. This was followed by the genetic aspect of the study that examined the influence of vitamin D receptor polymorphisms on biochemical markers of mineral bone disorders. Lastly, the study also evaluated the association between markers of CKD-MBD and mortality in MHD patients. Results The prevalence of hyperparathyroidism (iPTH>150 pg/mL), hyperphosphataemia, hypocalcaemia and 25-hydroxyvitamin D deficiency (<30 ng/mL) was 73.4%, 57.0%, 20.3% and 80.7 % respectively in our MHD patients. The combination of markers of bone turnover (iPTH>150 pg/mL and total alkaline phosphatase > 112 U/L) suggestive of high turnover bone disease, was present in 47.3 % of the study population. The odds ratios for developing secondary hyperparathyroidism with hypocalcaemia and hyperphosphataemia were 5.32 (95% CI 1.10 - 25.9, P =0.03) and 3.06 (95 % CI 1.15 - 8.10, P =0.02) respectively. The 293 CKD patients (208 blacks, 85 whites) had an overall mean age of 51.1±13.6 years, and black patients were significantly younger than the white patients (48.4 ±.13.6 versus 57.1±15.5 years; p<0.001). In comparison to whites, blacks had higher median iPTH (498 [37-1084] versus 274[131-595] pg/ml; P=0.03), alkaline phosphatase (122[89-192] versus 103[74-144] U/L; P=0.03) and mean 25- hydroxyvitamin D (26.8±12.7 versus 22.7 ±12.2 ng/ml, P=0.01) levels, while their median FGF23 (100 [34-639] versus 233[80-1370] pg/ml; P=0.002) and mean serum phosphate (1.3±0.5 versus 1.5±0.5, P =0.001) levels were significantly lower. With the exception of vitamin D receptor (VDR) Taq I polymorphism, the distribution of the VDR polymorphisms differs significantly between blacks and whites. In hemodialysis patients, the BsmI Bb genotype was significantly associated with moderate secondary hyperparathyroidism (OR, 3.88; 95 CI 1.13-13.25, P=0.03) and severe hyperparathyroidism (OR, 2.54; 95 CI 1.08-5.96, P=0.03). Patients with high total alkaline phosphatase (TAP) had significantly higher risk of death compared to patients with TAP <112 U/L (hazard ratio, 2.50; 95% CI 1.24–5.01, P = 0.01). Similarly, serum calcium >2.75 mmol/L was associated with increased risk of death compared to patients within levels of 2.10–2.37 mmol/L (HR 6.34, 95% CI 1.40–28.76; P = 0.02). The HR for death in white patients compared to black patients was 6.88; 95% CI 1.82–25.88; P = 0.004. Conclusions Secondary hyperparathyroidism and 25–hydroxyvitamin D deficiency were common in our haemodialysis patients. The study also highlighted the existence of racial differences in the circulating markers of mineral bone disorders in our African CKD population. In addition, the study showed that both moderate and severe secondary hyperparathyroidism are predicted by the BsmI Bb genotype, and the over expression of this genotype in black patients may partly explain the ethnic variations in the severity of secondary hyperparathyroidism in the CKD population. High levels of serum alkaline phosphatase, hypercalcaemia, and white race are associated with increased risk of death in MHD patients.
LG2018

碩士
國立成功大學
體育健康與休閒研究所
101
Purpose: To investigate the effects of different exercise training on muscular strength, risk factors of cardiovascular diseases, adipocytokines, and bone turnover markers. Method: Fifty-three middle-aged and elderly volunteers completed the study. They were randomly paired into four groups: the high-intensity resistance (HIR) (n=13, age 61.8±3.4 yrs; BMI 23.3±3.7 kg/m2), moderate-intensity resistance (MIR, n=14, age 61.4±5.2 yrs, BMI 23.7±1.8 kg/m2), multi-component exercise (MCE, n=13; age 61.7±4.5 yrs; BMI 24.5±3.4 kg/m2), and control groups (CON, n=13; age 61.5±4.4 yrs, BMI 24.9±5.4 kg/m2) by each subject’s mean of upper and lower maximal strength. Subjects in all the training groups engaged in exercise training twice per week under supervision for 24 weeks. Data collected at pre- and post-training included body fat mass, body lean mass, one repetition-maximum (1-RM), triglycerides, total cholesterol, high-density lipoprotein cholesterol, high-density lipoprotein cholesterol, adipocytokines (including adiponectin and leptin), bone mineral density (BMD), osteocalcin and cross-linked carboxy-terminal telopeptide of type I collagen of bone metabolic markers. Results: All of the experimental groups showed significant improvements in 1-RM compared with the CON (p 〈0.001), while the CON showed decreased muscular strength. The MCE had a significant increase in all blood lipids profiles and fasting insulin compared to other three groups (p 〈0.05). The HIR had a greater increase in BMD (+0.9%) than the other three groups (p 〈0.05). Adipocytokines and bone metabolic markers had no significant changes between groups (p 〉0.05). Only the MCE showed a significant decrease in the percentage of fat mass and trunk and total fat mass compared with the MIR (p〈0.05). All experimental groups had significantly augmented lean mass of the arm compared to the CON (p〈0.05). Conclusions: High- or medium intensity RT and multi-component exercise have positive and same effects on muscular strength and arm lean mass. Therefore, resistance training or multi-component exercise might decrease the bone turnover rate. High-intensity RT can further improve BMD due to higher stimulation. Multi-component exercise can elevate HDL-C and decrease fat present, trunk and total fat mass and perhaps decrease the risk of cardiovascular disease via aerobic training due to consecutive effects.