3种体外培养方法诱导骨髓间充质干细胞向心肌细胞分化效果比较

Comparison of differentiation effects of BMSCs into cardiomyocytes induced by three culture methods in vitro

目的:采用3种体外培养方法对骨髓间充质干细胞(BMSCs)进行诱导分化,探讨体外诱导BMSCs向心肌细胞分化的最佳方法。方法:采用密度梯度离心法结合贴壁培养法体外分离培养大鼠BMSCs,分为5-氮胞苷(5-aza)组、扩张型心肌病(DCM)模型大鼠心肌细胞裂解液组和DCM模型大鼠血清+5-aza组,同时设对照组(同等条件下在含10%胎牛血清的L-DMEM培养基中培养),观察细胞形态表现,采用RT-PCR法、Western blotting法和免疫组织化学染色法检测细胞中肌钙蛋白T (cTnT)的表达。结果:密度梯度离心法结合贴壁培养法获得大鼠BMSCs,BMSCs高表达间充质干细胞(MSCs)的特征性表面标记CD44、CD29和CD105,不表达造血干细胞的特征性表面标志CD34;特定诱导条件下,BMSCs可以向成骨细胞及脂肪样细胞分化。体外3种培养方法均可诱导BMSCs表达cTnT,分化为心肌样细胞;5-aza组细胞生长状态差,其余2组细胞生长状态较好,DCM模型大鼠血清+5-aza组细胞中cTnT阳性表达率最高。结论:采用DCM模型大鼠血清联合5-aza诱导BMSCs向心肌细胞分化的效果最佳。

Objective:To induce the differentiation of bone marrow mesenchymal stem cells(BMSCs)by three culture methods in vitro,and to explore the best method of inducing the BMSCs to differentiate into the cardiomyocytes in vitro.Methods:The rat BMSCs were separated and cultured with density gradient centrifugation combined with adherence culture method.The rat BMSCs were cultured with 5-azacytidine(5-aza),the myocardial cell cleavage of the dilated cardiomyopathy(DCM)model rats and the serum of DCM model rats+5-azain vitro,respectively;control group was set up at the same time(The BMSCs were cultured in the L-DMEM medium containing 10% FBS under the same condition).The morphology of BMSCs were observed and the expression of troponin T(cTnT)was detected by RT-PCR,Western blotting method and immunohistochemical staining.Results:The rat BMSCs were obtained by density gradient centrifugation combined with adherence culture method.The BMSCs highly expressed the characteristic surface marker CD44,CD29 and CD105 of mesenchymal stem cells(MSCs),and did not express the characteristic surface marker of hematopoietic stem cells CD34.Under the specific induction conditions,the BMSCs were differentiated into the osteoblasts and the aliphatic cells.In vitro,three culture methods could induce the BMSCs to express cTnT and differentiate into the cardiomyoid cells.In 5-aza group,the cell growth status was poor,and the cells in the other two groups had better growth status.The positive expression rate of cTnT was the highest in DCM model rat serum +5-aza group.Conclusion:The effect of DCM model rat serum combined with 5-aza to induce the BMSCs to differentiation into the myocardial cells is the best.