微环境中人骨髓间充质干细胞向心肌细胞表型分化的实验研究

Human bone marrow mesenchymal stem cells cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes differentiated into cardiomyocyte phenotype

目的探讨人骨髓间充质干细胞（hBMSC）在非接触共培养的微环境中向心肌细胞分化的能力。方法密度梯度离心法分离培养hBMSC，用流式细胞仪鉴别其表面标记特征。采用transwell按1：10的比例共同培养hBMSC和SD乳鼠心室肌细胞。用聚合酶链反应检测心肌转录因子GATA4的基因表达水平。免疫细胞化学、免疫荧光检测横纹肌仅一辅肌动蛋白、结蛋白、肌钙蛋白（cTn）T和cTnI等心肌细胞标志蛋白的表达。结果hBMSC的标志物主要为CD29（98．64％±0．80％）和CD44（96．70％±1．50％），而极少表达CD105（2．21％±0．600k）、CD11b（0．80％±0．05％）、CD45（1．39％±0．13％）和CD34（1．73％±0．08％）。与心肌细胞共培养后第7天可在hBMSC细胞中观察到少量搏动的细胞，随着诱导时间的增加，细胞的搏动更规则、有力。与心肌发育有关的转录因子GATA4基因在诱导后1、2、3周均有表达。共培养后2周，检测的心肌标志蛋白均有阳性表达，分别是结蛋白，仅一辅肌动蛋白，cTnI和cTnT。荧光标记也检测到cTnT和cTnI的阳性表达。结论hBMSC具有向心肌细胞分化的潜能，在共培养的微环境中，可以分化成心肌细胞表型，其机制是通过心肌细胞的旁分泌作用，而不需要hBMSC与心肌细胞的直接接触。

Objective To investigate the ability of human bone marrow mesenchymal stem cells （hBMSCs）, cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes, to differentiate into cardiomyocytes. Methods hBMSCs were isolated and purified by density gradient centrifugation and adherence screening method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer, hBMSCs were cocuhured with neonatal rat ventricular myocytes in a rate of 1：10 separated by semi-permeable membrane. GATA4 mRNA was detected by RT-PCR; Immunocytochemistry, and Immunostaining were used to detect sarcomeric α-actinin, desmin, cTnT, and cTnI protein level. Results CD29 （ 98. 64% ± 0. 80% ） and CD44 （96. 70% ± 1.50% ） were the major surface markers of hBMSCs. After coculturing with semi-permeable membrane separated neonatal rat ventricular myocytes, the first contraction of single cells was noted at day 7 and GATA4 expression was detected on these cells by RT-PCR after 1 to 3 weeks coculture. Desmin, sacomeric α-actinin, cTnI and cTnT could be detected by immunocytochemistry and immunostaining on some of these cells. Conclusion hBMSCs possess the potential to differentiate into myocardial cell phenotype in the cardiac microenvironment. Direct contact with cardiomyocytes was not necessary required for hBMSCs differentiation.