Implantação da técnica para diferenciação osteoblástica a partir de células tronco mesenquimais humanas derivadas do tecido adiposo
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Data
2013-02-26
Autores
Olimpio, Regiane Marques Castro [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Introdução: As Células Tronco Mesenquimais (CTMs) são capazes de diferenciar em várias linhagens de células. As CTMs, quando em cultura, e na presença de citocinas ativadoras de osso, sofrem osteoindução e diferenciam em osteoblastos. Entretanto, pouco se conhece sobre a função e a proliferação dos progenitores osteoblásticos, que pode ser essencial para a integridade do tecido ósseo. Objetivo: Implantar a técnica para diferenciação osteoblástica a partir de células tronco mesenquimais humanas derivadas do tecido adiposo. Metodologia: O tecido adiposo foi obtido de três pacientes do sexo feminino submetidas à abdominoplastia. As CTMs foram mantidas em meio D-MEM completo contendo 10% de SFB, mantidas a 37° C em uma atmosfera umidificada com 95% O2/5% CO2. Após a confluência alíquotas de células foram separadas para o processo de caracterização celular. Após esse período, foi realizada osteoindução utilizando meio DMEN completo com acréscimo de dexametasona, ácido ascórbico e βglicerofosfato. As células foram mantidas nesse meio por até 16 dias. Como controle negativo da indução, culturas de células foram mantidas em meio convencional pelo mesmo período de tempo. Ao término de 16 dias as células foram separadas para a caracterização celular e o RNA foi coletado para análise da expressão de RANKL. Resultados: As células após seis dias de cultura apresentaram aderidas com 40% de confluência e após dezessete dias com 75% de confluência. As CTMs expressaram anticorpo CD 44 e 90. Foi encontrado médio nível (20%- 40%) de dano de DNA em 40% das CTMs analisadas. A imunofluorescência confirmou a homogeneidade e positividade para expressão de anti-vimentina. A diferenciação química foi conseguida pela adição de dexametasona, ácido ascórbio e βglicerofosfato ao meio DMEN completo. Houve a expressão de...
Mesenchymal Stem Cells (MSCs) are able to differentiate into multiple cell lineages. The MSCs, when cultured, in the presence of activating cytokines bone suffer osteoinduction and differentiate into osteoblasts. However, little is known about the function and proliferation of osteoblastic progenitors, which can be essential for the integrity of bone tissue. Objective: Deploy the technique for osteoblast differentiation from human mesenchymal stem cells derived from adipose tissue. Methods: Adipose tissue was obtained from three female patients undergoing abdominoplasty. MSCs were maintained in complete D-MEM containing 10% FBS, maintained at 37 ° C in a humidified atmosphere with 95% O2 / 5% CO2. After confluence aliquots of cells were separated into the cellular characterization process. After this period, was performed using osteoinduction medium DMEN complete with addition of dexamethasone, ascorbic acid and βglicerofosfato. Cells were maintained in this medium for up to 16 days. As a negative control induction, cell cultures were maintained in standard medium for the same period of time. At the end of 16 days the cells were separated for the characterization and cellular RNA was collected for analysis of expression of RANKL. Results: The cells after six days of culture had adhered with 40% confluency and after seventeen days with 75% confluency. The MSCs expressed antibody CD 44 and 90. Found average level (20% - 40%) of DNA damage in 40% of MSCs analyzed. Immunofluorescence confirmed the homogeneity and positivity for anti-vimentin expression. The chemical differentiation was achieved by addition of dexamethasone, ascórbio acid and βglicerofosfato DMEN in half full. There expression of alkaline phosphatase, osteocalcin, RANKL and mineralized matrix formation from osteoblasts after 16 days with osteogenic... (Complete abstract click electronic access below)
Mesenchymal Stem Cells (MSCs) are able to differentiate into multiple cell lineages. The MSCs, when cultured, in the presence of activating cytokines bone suffer osteoinduction and differentiate into osteoblasts. However, little is known about the function and proliferation of osteoblastic progenitors, which can be essential for the integrity of bone tissue. Objective: Deploy the technique for osteoblast differentiation from human mesenchymal stem cells derived from adipose tissue. Methods: Adipose tissue was obtained from three female patients undergoing abdominoplasty. MSCs were maintained in complete D-MEM containing 10% FBS, maintained at 37 ° C in a humidified atmosphere with 95% O2 / 5% CO2. After confluence aliquots of cells were separated into the cellular characterization process. After this period, was performed using osteoinduction medium DMEN complete with addition of dexamethasone, ascorbic acid and βglicerofosfato. Cells were maintained in this medium for up to 16 days. As a negative control induction, cell cultures were maintained in standard medium for the same period of time. At the end of 16 days the cells were separated for the characterization and cellular RNA was collected for analysis of expression of RANKL. Results: The cells after six days of culture had adhered with 40% confluency and after seventeen days with 75% confluency. The MSCs expressed antibody CD 44 and 90. Found average level (20% - 40%) of DNA damage in 40% of MSCs analyzed. Immunofluorescence confirmed the homogeneity and positivity for anti-vimentin expression. The chemical differentiation was achieved by addition of dexamethasone, ascórbio acid and βglicerofosfato DMEN in half full. There expression of alkaline phosphatase, osteocalcin, RANKL and mineralized matrix formation from osteoblasts after 16 days with osteogenic... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Células-Tronco, Tecido adiposo, Matriz extracelular, Ossos - Crescimento, Stem cells
Como citar
OLIMPIO, Regiane Marques Castro. Implantação da técnica para diferenciação osteoblástica a partir de células tronco mesenquimais humanas derivadas do tecido adiposo. 2013. 58 f. Dissertação (mestrado) - Universidade Estadual Paulista Julio de Mesquita Filho, Faculdade de Medicina de Botucatu, 2013.