电刺激促骨髓间充质干细胞向心肌样细胞分化

Effects of Electrical Stimulation on the Differentiation of Mesenchymal Stem Cells into Cardiomyocyte-like Cells

观察体外电刺激对大鼠骨髓间充质干细胞(rBMSCs)向心肌样细胞分化的影响。选用第3、4代rBMSCs用于实验。实验分为3组:(1)电刺激组,rBMSCs分别暴露于1、2、4、6V电压,频率2Hz,脉宽5ms的电刺激,2h/d,连续刺激10d;(2)5-氮胞苷(5-Aza)组,给予10μmol/L 5-Aza诱导24h,然后采用完全培养液培养10d;(3)对照组,采用完全培养液培养10d。通过倒置相差显微镜逐日连续观察细胞的生长状况和形态特征;采用实时荧光定量PCR方法检测GATA4,α-肌动蛋白(α-actin),α-辅肌动蛋白(ACTN2),心肌肌钙蛋白T(TNNT2)的mRNA表达;采用免疫荧光染色技术检测TNNT2的蛋白表达。结果显示:电刺激组、5-Aza组的GATA4、α-actin、ACTN2和TNNT2mRNA表达与TNNT2蛋白表达明显高于对照组(P<0.05);提示,电刺激可以促进rBMSCs向心肌样细胞分化,电压2V的电刺激效果最佳。

The aim of this study is to investigate the effects of electrical stimulation （ES） on the induction of rat bone marrow mesenchymal stem cells （rBMSCs） to differentiate into cardiomyocyte like cells in vitro. The third or fourth generation of the rBMSCs was randomly divided into three groups, i.e. ES group, 5-Azacytidine （5-Aza） group, and control group. Those in the ES group with complete medium were exposed to 1,2,4 and 6V, 2Hz, 5ms ES for 2h everyday,lasting for 10d. Those in the 5-Aza group were induced by 10 9mol/L 5-Aza for 24h, then the medium was changed to complete medium without 5-Aza. Those in the control group were only cultured with complete medium. The growth status and morphological features of rBMSCs were observed by inverted phase microscope. The mRNA expressions of GATA4, a-actin, ACTN2 and TNNT2 were determined by Real-time fluorescent quantification PCR, and the protein expression of TNNT2 was detected with immunoftuorescence staining. The results showed that the mRNA expression level of the GATA4, a-actin, ACTN2 and TNNT2 and the protein expression level of the TNNT2 were significantly higher in the ES group and 5-Aza group, compared to those in the control group（P〈0.05）. It suggested that ES could induce rBMSCs differentiation into cardiomyocyte-like cells in vitro.