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目的:探讨羰基-氰-对-三氟甲氧基本腙(carbonyl cyanide p-trifluoromethoxyphenylhydrazone,FCCP)对P19细胞向心肌细胞诱导分化的影响。方法:在P19细胞向心肌细胞诱导分化的过程中加入5μmol/L的FCCP,同时设立阴性对照组,显微镜观察分化过程中不同时间点P19细胞形态的变化,并采用real-time PCR和蛋白质印迹法分别检测GATA4、NKX2.5及cTnT在分化过程中的mRNA和蛋白表达水平。结果:FCCP处理组P19细胞形成的胚胎样小体较对照组体积小,结构较松散,形态不均一。在整个分化过程中,FCCP处理组GATA4、NKX2.5及cTnT基因均有表达,但整个动态表达过程较对照组延迟;Western blot结果显示,FCCP处理组GATA4、NKX2.5蛋白在第5、7、9天的表达量均较对照组低;在第11天,两组间GATA4蛋白的表达量差异无统计学意义;而NKX2.5在FCCP处理组的表达量仍低于对照组。FCCP处理组和对照组cTnT蛋白在分化第5、7天的表达量差异无统计学意义,而在第9、11天cTnT蛋白表达量FCCP组均较对照组低。结论:FCCP可抑制P19细胞向心肌细胞诱导分化的进程。
Objective: To investigate the effect of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on the differentiation of P19 cells into cardiomyocytes. Methods: FCM of 5μmol / L was added during the differentiation of P19 cells into cardiomyocytes. At the same time, a negative control group was set up. The morphological changes of P19 cells at different time points during the differentiation were observed by microscope. Real-time PCR and Western blot The mRNA and protein expression levels of GATA4, NKX2.5 and cTnT during differentiation were detected respectively. Results: The volume of embryoid bodies formed by P19 cells in FCCP-treated group was smaller than that in control group, with loose structure and heterogeneous morphology. During the whole process of differentiation, GATA4, NKX2.5 and cTnT genes were all expressed in FCCP-treated group, but the whole dynamic process was delayed compared with that in control group. Western blot results showed that FCCP- , And the expression of GATA4 protein was lower than that of the control group on the 9th day. On the 11th day, there was no significant difference in the expression of GATA4 protein between the two groups; however, the expression level of NKX2.5 in FCCP group was still lower than that in the control group. There was no significant difference in the expression of cTnT protein between the FCCP group and the control group on day 5 and 7, while the expression of cTnT protein in FCCP group was lower than that of control group on the 9th and 11th day. Conclusion: FCCP can inhibit the differentiation of P19 cells into cardiomyocytes.