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目的探讨用5-氮胞苷(5-aza)体外诱导骨髓间充质干细胞(MSCs)分化为心肌样细胞的最佳条件。方法用Percoll密度梯度离心法分离培养、纯化大鼠MSCs,取第3代MSCs随机分为A、B、C 3组,分别加入5、10、20μm ol/L5-aza;每组分别孵育12、24、48 h,继续培养28 d。相差显微镜观察细胞形态变化,免疫组化法检测细胞肌动蛋白(α-ac-tin)和心肌肌钙蛋白T(cTnT)表达,并通过W estern印迹法对细胞cTnT进行定量分析。结果MSCs经5-aza诱导培养28 d后,电镜下见细胞胞浆有散在肌丝样结构,免疫组化检测α-actin和cTnT表达均呈阳性;5μm ol/L的5-aza孵育48 h、10μm ol/L孵育24 h培养28 d的细胞cTnT表达量与10μm ol/L的5-aza孵育48 h、20μm ol/L 3种孵育时间各组比较无显著性差异,且细胞生物学特性未受到明显影响。结论MSCs在体外经5-aza诱导后可分化为心肌样细胞;5μm ol/L的5-aza孵育48 h和10μm ol/L的5-aza诱导24 h是MSCs体外诱导分化心肌样细胞的理想条件。
Objective To investigate the optimal conditions for the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte-like cells induced by 5-azacytidine (5-aza) in vitro. METHODS: Rat MSCs were isolated and cultured using Percoll density gradient centrifugation. MSCs of the third generation were randomly divided into A, B and C groups, and were respectively treated with 5, 10, and 20μmol / L 5-aza. 24,48 h, continue to cultivate 28 d. The morphological changes of cells were observed by phase contrast microscope. The expression of actin (α-ac-tin) and cardiac troponin T (cTnT) was detected by immunohistochemistry. The cTnT was quantified by Western blotting. Results MSCs were cultured in vitro after 5-aza induction for 28 days, and the cytoplasm of the MSCs were observed under electron microscope. The positive expression of α-actin and cTnT was detected by immunohistochemistry. After 5-ol / L 5-aza treatment for 48 h , The expression of cTnT in cells incubated with 10μm ol / L for 24 h was not significantly different from that in cells incubated with 10μmol / L 5-aza for 48 h and 20μm ol / L, and the cell biological characteristics Has not been significantly affected. Conclusion MSCs can differentiate into cardiomyocyte-like cells induced by 5-aza in vitro. Incubation of 5-aza with 5μmol / L for 48 h and 10μmol / L of 5-aza for 24 h is the ideal method for MSCs to differentiate into cardiomyocytes in vitro condition.