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  

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

作　　者：张彐宁 高维娟[1] 周晓红[1] 靳晓飞(指导) ZHANG Xue-Ning;GAO Wei-Juan;ZHOU Xiao-Hong;JIN Xiao-Fei(Hebei University of Chinese Medicine,Hebei Key Laboratory of Chinese Medicine Research on Cardiocerebrovascular Disease,Shijiazhuang 050091,China)

机构地区：[1]河北中医学院,河北省心脑血管病中医药防治研究重点实验室,石家庄050091

出　　处：《中国免疫学杂志》2021年第1期21-25,30,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金面上项目(81873180);河北省教育厅青年基金项目(QN2018059);河北省中医药管理局科研计划项目(2019093);河北省研究生创新资助项目(CXZZSS2019066);河北中医学院优秀青年教师基础研究计划项目(YQ2018005);河北中医学院青年教师科研基金项目(QNZ2018001);河北中医学院2019年省属高校基本科研业务费项目(YXZ2019012);河北省中医药管理局科研计划项目(2021103)。

摘　　要：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。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

关 键 词：APAF-1 凋亡 线粒体凋亡通路 PC12细胞 

分 类 号：R363[医药卫生—病理学] R392.3[医药卫生—基础医学]