体外电刺激诱导大鼠骨髓间充质干细胞心肌特异性标志物MEF-2C和Cx43的表达

Expression of Myocardial Specificity Markers MEF-2Cand Cx43in Rat Bone Marrow-derived Mesenchymal Stem Cells Induced by Electrical Stimulation In Vitro

骨髓间充质干细胞(BMSCs)由于具有向心肌方向分化的潜能,目前已成为一种新的能够修复心脏组织损伤的重要治疗选择。在本实验中我们探讨了体外不同的电刺激时间对BMSCs向心肌方向分化的影响。实验共分为三组:1电刺激组,完全培养基培养的大鼠骨髓间充质干细胞(rBMSCs)受到电压为2V,频率为2 Hz,脉宽为5ms的电刺激,作用时间为30min/d、2h/d、4h/d、6h/d,连续作用10d;25-氮胞苷(5-Aza)组,5-Aza(10μmol/L)诱导rBMSCs 24h,然后采用完全培养基培养10d;3空白对照组,完全培养基静态培养rBMSCs 10d。通过实时荧光定量PCR方法检测各组rBMSCs的肌细胞特异性增强因子-2C(MEF-2C)mRNA和缝隙连接蛋白43(Cx43)mRNA的表达;通过Western blot方法检测各组rBMSCs的MEF-2C蛋白和Cx43蛋白的表达。实验结果显示:不同持续时间的电刺激组及5-Aza组的MEF-2CmRNA、Cx43mRNA表达量及MEF-2C蛋白、Cx43蛋白表达量均明显高于空白对照组(P<0.05)。本实验结果提示,体外电刺激可以诱导rBMSCs向心肌方向分化,其中2h/d作用效果最佳,但其机制尚未明确。

Bone marrow-derived mesenchymal stem cells（BMSCs）for repairing damaged heart tissue are a new kind of important treatment options because of their potential to differentiate into cardiomyocytes.We in this experiment investigated the effect of different electrical stimulation time on the expression of myocardial specificity gene and protein in rat bone marrow mesenchymal stem cells（rBMSCs）in vitro.The rBMSCs of second or third generation were randomly divided into three groups,i.e.electrical stimulation（ES）group,5-Azacytidine（5-Aza）group and the control group.The rBMSCs in the ES groups with complete medium were exposed to 2V,2Hz,5ms electrical stimulation for 0.5h,2h,4h,and 6hrespectively every day for 10 days.Those in the 5-Aza group were induced by 5-Aza（10μmol/L）for 24 h,and then cultured with complete medium for 10 days.Those in the control group were only cultured with complete medium,without any treatment,for 10 days.The rBMSCs＇ morphological feature in each group was observed with inverted phase microscope.The mRNA expression of myocyte-specific enhancer factor 2C（MEF-2C）and connexin 43（Cx43）were examined with Real-Time quantitative PCR and the protein expression of MEF-2C,Cx43 were detected with Western Blot method.The results showed that the mRNA expression level of the MEF-2C,Cx43 and the protein expression level of MEF-2C,Cx43 were significantly higher in the ES group and 5-Aza group than those in the relative control group（P0.05）.It suggests that electrical stimulation could play apart of role in the induction of the rBMSCs to differentiate into the cariomyocyte-like cells in vitro and the effectiveness of the electrical stimulation with 2h/d had the best in our experiement.But the mechanism how electrical stimulation promotes the differentiation of rBMSC into cardiomyocyte is still unclear.