沉默Apaf-1 基因对氧糖剥夺/ 复氧复糖PC12 细胞线粒体凋亡通路的影响

目的:探讨RNA干扰沉默Apaf-1基因对氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路的影响。方法:PC12细胞随机分为3组:正常组(Control)、模型组(Model)、Apaf-1基因沉默组(Apaf-1-siRNA)。正常组于CO2培养箱内正常培养,其余2组给予氧糖剥夺2 h、复氧复糖24 h处理,Apaf-1-siRNA组于造模前将化学合成的siRNA通过脂质体转染于PC12细胞靶向沉默Apaf-1基因。用荧光标记的siRNA检测Apaf-1转染效率,Western blot检测转染后PC12细胞Apaf-1蛋白表达,CCK-8检测细胞存活率,TUNEL染色检测细胞凋亡指数,流式细胞术检测细胞凋亡率,免疫荧光染色检测Bax/Bcl-2比值,Western blot检测线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达。结果:Apaf-1-siRNA可有效沉默PC12细胞Apaf-1蛋白表达(P<0.05)。与Control组相比,Model组细胞存活率明显降低(P<0.05),细胞凋亡指数和凋亡率显著升高(P<0.05),Bax/Bcl-2比值升高(P<0.05),线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达显著升高(P<0.05);与Model组相比,Apaf-1-siRNA组细胞存活率显著升高(P<0.05),细胞凋亡指数和凋亡率显著降低(P<0.05),Bax/Bcl-2比值降低(P<0.05),Apaf-1、caspase-9、caspase-3蛋白表达均明显降低(P<0.05)。结论:靶向沉默Apaf-1基因可有效降低氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达,抑制细胞凋亡,提高细胞存活率。

Effects of Apaf-1 gene silencing on mitochondrial apoptotic pathway in oxygen-glucose deprivation/reoxygenation PC12 cells

Objective:To investigate effect of RNA interference silencing Apaf-1 gene on mitochondrial apoptosis pathway in oxygen-glucose deprivation/reoxygenation PC12 cells.Methods:PC12 cells were randomly divided into three groups:Control group,Model group and Apaf-1 gene silencing group(Apaf-1-siRNA).Control group was cultured in CO2 incubator,and other groups were treated with oxygen-glucose deprivation for 2 h,reoxygenation for 24 h,Apaf-1-siRNA group transfected chemically synthesized siRNA into PC12 cells by liposome before modeling for targeting and silencing Apaf-1 gene.Transfection efficiency of Apaf-1 gene was observed by immunofluorescence.Western blot was used to detect expression of Apaf-1 protein in PC12 cells after transfection.CCK-8 was used to detect cell survival rate.TUNEL staining was used to detect apoptotic index of each group.Flow cytometry was used to detect apoptotic rate.Immunofluorescence staining was used to detect Bax/Bcl-2 ratio.Western blot was used to detect apoptotic pathway of mitochondria diameter-critical proteins expressions of Apaf-1,caspase-9 and caspase-3.Results:Apaf-1-siRNA could effectively silence expression of Apaf-1 protein in PC12 cells(P<0.05).Compared with control group,cell survival rate in model group was significantly decreased(P<0.05),and apoptotic index and apoptotic rate were significantly increased(P<0.05),ratio of Bax/Bcl-2 was significantly increased(P<0.05),and expressions of key mitochondrial apoptotic pathway proteins Apaf-1,caspase-9 and caspase-3 were significantly increased(P<0.05).Compared with Model group,cell survival rate in Apaf-1-siRNA group was significantly increased(P<0.05),cell apoptosis rate was significantly decreased(P<0.05),Bax/Bcl-2 ratio was significantly decreased(P<0.05),and expressions of key mitochondrial apoptotic pathway proteins Apaf-1,caspase-9 and caspase-3 were significantly decreased(P<0.05).Conclusion:Targeted silencing of Apaf-1 gene can effectively reduce expressions of key proteins Apaf-1,caspase-9 and caspase-3 in mitochondrial apoptotic pathway of oxygen-glucose deprivation/reoxygenation PC12 cells,inhibit cell apoptosis and improve cell survival rate.