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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 study the changes of DNA methylation level in promoter region of Nkx2.5 gene in early development of myocardium with Islet-1 induced by directional differentiation of C3H10T1 / 2 cells into cardiomyocytes. Methods: The lentiviral vector expressing Islet-1 gene was transfected into C3H10T1 / 2 mesenchymal stem cells. The expression of Islet-1 was detected by immunofluorescence. At the time when Nkx2.5 reached its peak, the expression of Islet-1 was detected by methylation specific PCR The level of DNA methylation in the promoter region was detected. The expression of DNA methylation in C3H10 group (blank control group), negative control group (lentiviral empty vector) and experimental group (lentiviral vector transfected with Islet-1 gene) Promoter region DNA methylation levels. Results: The DNA methylation level of Nkx2.5 promoter in experimental group was lower than that in other two groups (all P <0.05). CONCLUSION: During the differentiation of C3H10T1 / 2 cells into cardiomyocytes, the expression level of DNA methylation in promoter region of early development-related genes is decreased and the expression of Islet-1 is promoted.