5-氮胞苷体外诱导骨髓间充质干细胞向心肌样细胞的分化

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(MSC)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MSC,用流式细胞仪检测细胞表面抗原。以5-Aza诱导第3代MSC 24 h后继续培养。培养4周,用免疫细胞化学染色法检测肌系标记抗原:α-肌动蛋白(α-actin)及心肌细胞特异性标记抗原:肌钙蛋白T(cTnT);在透射电镜下观察细胞的超微结构。结果:MSC经5-Aza诱导分化后,可表达α-actin和cTnT,未经诱导的同培养天数的MSC中均未见表达。透射电镜可观察到肌丝等心肌细胞的特异性结构。结论:5-Aza可诱导MSC分化为心肌样细胞。

AIM： To observe the effect of 5-azacytidine （5-Aza） on bone marrow mesenchymal stem cells （MSCs） and to determine the cardiomyocyte-like cells that were differentiated from MSCs. METHODS： Bone marrow mononuclear cells （BM-MNC） were isolated from bone marrow by the method of density gradient centrifugation and were cultured in low glucose DMEM containing 200 ml/L fetal bovine serum. The cells were purified by the different abilities of adhesion. Cell surface antigens were tested by flow cytometry. MSCs were induced by 5-Aza at the third passage for 24 h. After being treated by 5-Aza for 4 weeks, the cells were harvested. Muscle tissue antigen such as α-actin and the cardiac muscle heterologous protein, e.g., cTnT, were detected by immunocytochemical staining. Ultramicrostructure of the cells was observed under transmission electron microscope. RESULTS： After being induced by 5-Aza, α-actin and cTnT were expressed in MSCs. However, expression in MSC without 5-Aza treatment was negative. Ultramicrostructure of the cells such as myofilament was observed under transmission electron microscope. CONCLUSION： Human bone marrow MSCs can differentiate into cardiomyocyte-like cells under 5-Aza induction in vitro.