骨髓间充质干细胞向心肌样细胞分化的实验研究

The differentiation of human mesenchymal stem cells into cardiomyocyte-like cells in vitro induced by 5-azacytidine

目的研究5-氮胞苷(5-Aza)对培养人骨髓间充质干细胞(MSCs)的作用,并对分化后的心肌样细胞进行鉴定。方法采用密度梯度离心法分离到骨髓单个核细胞(MB-MNC),用含200 ml/L胎牛血清的低糖型DMEM培养液进行培养。采用差速贴壁法纯化MSCs,用流式细胞仪检测细胞表面抗原。以5-Aza诱导第3代MSCs 24 h后,继续培养。于培养的第4周,采用免疫细胞化学方法检测肌系特异性标记抗原结合蛋白(Desmin),RT-PCR检测心肌早期转录因子GATA-4和心肌特异性蛋白肌钙蛋白T(cTnT)。结果MSCs经5-Aza诱导分化后表达Desmin、GATA-4和cTnT,未经诱导的同培养天数的MSCs中均呈未见表达。结论MSCs经5-Aza诱导后可以表现心肌样细胞特征。

Objective To observe the effect of 5-azacytidine（5-Aza） on bone marrow mesenchymal stem cells （MSCs） and accredit the cardiomyocyte - like cells which were differentiated from MSCs. Methods Bone marrow mononuclear cells（MB-MNC） were isolated from bone marrow by the density gradient centrifugation method and were cultured in low glucose DMEM containing 200 ml/L fetal bovine serum. The cells were purified by the different ability of adhesion.The cell surface antigens were tested by flow cytome try. MSCs were induced by 5-Aza at the third passage for 24 hours. After being treated by 5-Aza for 4 weeks,the cells were harvested. The antigen of muscle tissue such as Desmin were detected by immunocytochemical staining. The early transcription factor of cardiac muscle such as GATA-4 and the cardiac muscle heterologous protein e.g.cTnT were detected by RT-PCR.Results After being induced by 5- Aza, Desmin, GATA-4 and cTnT were expressed in MSCs; however, the expression in MSCs without 5 Aza treatment was negative. Conclusion Human bone marrow MSCs can diffentiate into cardiomyocyte-like cells under 5-Aza induction in vitro.