过表达Seipin对大鼠肾上腺嗜铬细胞瘤细胞凋亡的影响

Protective effect of seipin overexpression on rat pheochromocytoma cells

目的构建Seipin过表达PC12细胞株,探讨Seipin过表达后PC12细胞的凋亡情况。方法将PC12细胞分为正常组(未经任何处理的PC12细胞)、阴性组(不带Seipin过表达基因的慢病毒感染的PC12细胞株)和过表达组(带Seipin过表达基因的慢病毒感染的PC12细胞株);进行相应处理后培养24 h,采用荧光显微镜观察红色荧光基因(慢病毒携带的Seipin基因和空载体都包含了红色荧光基因)在细胞中的表达,采用Real time PCR检测BSCL2 mRNA表达水平,Western blot检测凋亡相关蛋白BCL2-Associated X蛋白质(Bax)的表达水平;TUNEL染色观察细胞内断裂DNA情况。结果正常组未观察到红色荧光,阴性组和过表达组均出现红色荧光,感染效率>80%,提示成功构建Seipin过表达的PC12细胞株;Real time PCR结果显示,过表达组BSCL2 mRNA水平相较正常组和阴性组升高,差异有统计学意义(P<0.05);Western blot结果显示,过表达组Seipin蛋白表达水平高于正常组和阴性组、凋亡相关蛋白Bax低于正常组和阴性组,差异有统计学意义(P<0.01),TUNEL染色观察到过表达组细胞核荧光强度弱于正常组和阴性组。结论Seipin可能对PC12细胞有保护作用。

Objective To investigate the effect of Seipin overexression on PC12 cell apoptosis via constructing PC12 cells overexpressing Seipin.Methods PC12 cells were treated without any as normal group,treated with lentivirus carrying empty vector as negative group,with lentivirus carrying Seipin gene as Seipin overexpression group.Lentivirus-carried Seipin vector or empty vector contain red fluorescent gene.At 24 hours after the treatment,the red fluorescence expression was observed with a fluorescence microscope.The expression level of BSCL2 mRNA was detected by Real time PCR,and the protein expression level of the apoptosis-related Bax was detected by Western blot.TUNEL staining was utilized to observe the morphology of apoptotic cells.Results No red fluorescence was observed in the normal group,while red fluorescence appeared in both the negative group and Seipin overexpression group.The infection efficiency was over 80%,indicating that PC12 cell line overexpressing Seipin was successfully constructed.Real time PCR results showed that the BSCL2 mRNA level was significantly increased in Seipin overexpression group compared with the normal group and the negative group(P<0.05).Western blot results showed that the Seipin protein expression level was the highest in Seipin overexpression group(P<0.05).The apoptosis related protein Bax was the lowest in Seipin overexpression group(P<0.01).TUNEL staining result showed that the nuclear fluorescence intensity was the most weak in Seipin overexpression group.Conclusion Seipin may have a protective effect on PC12 cells.