骨髓间充质干细胞向心肌分化的实验研究

Experimental study of mouse bone marrow mesenchymal stem cells induced to differentiate into the Cardiomyocyte-like cells

目的:研究骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)分化为心肌样细胞的能力,用于心肌补片治疗心肌梗死的研究。方法:分离C57/BSL小鼠BMSCs,全培养差速贴壁法,经过贴壁培养至第3代,流式细胞仪鉴定细胞表面标志(CD34、CD45、CD73、CD90),经10μmol/L的5-氮杂胞苷诱导细胞,24 h后更换完全培养基培养,2 w后进行免疫荧光染色,荧光显微镜观察心肌钙蛋白T(cTnT)和连接素蛋白43(CX43)的表达。结果:流式鉴定结果显示CD34、CD45阴性,CD73强阳性,CD90弱阳性。免疫荧光染色显示,诱导后细胞高表达心肌细胞特异性蛋白cTnT,连接素蛋白CX43表达水平明显增加。结论:5-aza可以诱导BMSCs大量表达心肌特异性蛋白cTnT和细胞连接素蛋白(CX43),干细胞分化为心肌样细胞,为干细胞移植治疗小鼠心梗提供种子细胞。

Objective：To study the ability of bone marrow-derived mesenchymal stem cells（MSC） to differentiate into cardiomyocyte-like cells for the treatment of myocardial infarction myocardial patch.Methods： separation C57/BSL mouse bone marrow-derived MSC and cultured differential adherence method,after adherent culture to the 3rd generation and flow cytometry to identify cell surface markers（CD34,CD45,CD73,CD90）,after 10μmol / L 5-azacytidine induced cell 24h after the replacement of complete medium culture 2W after immunofluorescence staining,fluorescence microscopy observation of cardiac troponin T（cTnT） and catenin protein 43（CX43） expression.Results： Flow Cytometry Analysis results show that CD73 strongly positive of CD90 weakly positive,CD34,CD45 negative.Immunofl uorescence staining showed that the cells expressed high induced cardiac myocyte specific protein cTnT,catenin protein CX43 expression levels were significantly increased.Conclusion：5-aza can induce a large number of bone marrow MSC express the cardiac-specific protein cTnT and cell connection the fibroin（CX43）,stem cells differentiate into cardiomyocyte-like cells,seed cells for stem cell transplantation in the treatment of mice with myocardial infarction.