Islet-1促MSCs心肌特化过程中Nkx2.5启动子区域DNA甲基化水平变化

DNA methylation of Nkx2.5 level during Islet-1 promotes cardiac specific differentiation of mesenchymal stem cells

目的:研究Islet-1诱导C3H10T1/2细胞定向分化为心肌样细胞过程中表达增加的心肌早期发育相关基因Nkx2.5启动子区域DNA甲基化水平的变化情况。方法:高表达Islet-1基因的慢病毒载体转染间充质干细胞C3H10T1/2,免疫荧光检测Islet-1表达情况,在Nkx2.5表达达高峰的时间点,通过甲基化特异性PCR技术检测其启动子区域DNA甲基化水平,比较C3H10组(空白对照组)、阴性对照组(转染慢病毒空载体)和实验组(转染高表达Islet-1基因的慢病毒载体)上述基因启动子区域的DNA甲基化水平。结果:实验组Nkx2.5基因启动子区域DNA甲基化水平低于其他两组(均P<0.05)。结论:高表达Islet-1促C3H10T1/2细胞特异性向心肌细胞方向分化过程中,心肌早期发育相关基因启动子区域DNA甲基化水平降低,促进其表达增加。

Objective：To investigate the DNA methylation changes of cardiac relative gene Nkx2. 5 during the process Islet-1 promotes cardiac specific differentiation of C3H10T1/2 cells. Method.. Lentiviral vectors that ex- pressed Islet-1 （Lenti-Islet-1） were constructed and used for C3H10T1/2 cells transduction. Flow cytometry was used to detect the transfection efficiency of C3H10T1/2 cells transfected with lentiviral vectors. Fluorescence mi- croscope was used to observe Islet-1 expression. Methylation specific PCR was used to detect the DNA methyla- tion differences of Nkx2.5 among C3H10 group, negative control group and experiment group. Result; DNA methylation of cardiac relative gene Nkx2.5 in experiment goup was much lower than that in C3H10 group and negative control group （both P〈0. 05）. Conclusion： DNA methylation of cardiac relative genes reduce to promote these genes expressions during the process mesenchymal stem cells are specific differentiated into cardiomyocyte- like cells.