5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌样细胞分化的实验研究

摘要

目的:研究体外培养大鼠骨髓间充质干细胞(mensenchymal stem cells,MSCs)经5-氮胞苷(5-aza)诱导定向分化为心肌样细胞形态学及分子生物学特征.方法:SD大鼠股骨骨髓经密度梯度离心及贴壁分离培养传代MSCs,体外用5-aza定向诱导向心肌样细胞分化.相差显微镜观察心肌样细胞形态学变化.免疫组化检测肌钙蛋白T(cTnT)、α-肌动蛋白和缝隙连接蛋白Cx43表达.结果:密度梯度离心及贴壁法分离培养的MSCs生长稳定,增殖快.5-aza诱导后,部分细胞形态发生变化,有肌管样结构形成随诱导后培养时间延长MSCs中cTnT、α-肌动蛋白和缝隙连接蛋白Cx43表达逐渐增强.结论:密度梯度离心及贴壁分离培养可获得大量纯度较高的骨髓间充质干细胞.经5-aza诱导骨髓间充质干细胞体外可分化为心肌样细胞.%Objective:To observe the cardiomyogenic potential and biological characteristics of mesenchymal stem cell (MSCs) induced by 5-azacytidine (5-aza) in vitro. Methods;MSCs were isolated from bone marrow of mice by density gradient centrifugation and attachment culture. MSCs of passage 2 were induced to differentiate by 5-aza at 10.0 μmol/L Optical microscope was used to observe the morphology of cells. cTnT.a-actin and connexin-43 were detected by immunochemistry to identify the differentiation. Results; MSCs isolated by density gradient centrifugation and attachment culture can grow steadily and proliferate rapidly. After incubated with 5-aza,MSCs were found to change their round shape into spindle shape and form myotube structure. cTnT,a-actin and connexin-43 expression was gradually up-regulated with the extension of culture time. Conclusion; The density gradient centrifugation and attachment culture method can be used for isolating MSCs with high purity. After treatment with 5-aza, MSCs can differentiate into cardiomyogenic cells with typical morphological and histological characteristic in vilro.