摘要
目的探讨线粒体外膜转位酶70(Tom70)/多核糖核苷酸核苷转移酶1(PNPT1)线粒体转位调控在大鼠心肌细胞缺氧性损伤中的作用及其机制。方法(1)将大鼠H9C2心肌细胞分为对照组(常氧条件培养12h)与缺氧组(缺氧干预12h),采用流式细胞术检测心肌细胞凋亡率,qPCR检测TUBA mRNA表达情况,Western blotting检测PNPT1蛋白表达情况。(2)利用携带Tom70序列的慢病毒载体转染H9C2细胞,分为NC组(慢病毒阴性对照组,转染慢病毒空载体)、Tom70过表达组(转染携带Tom70序列的慢病毒载体)、缺氧+NC组、缺氧+Tom70过表达组,采用Western blotting检测各组Tom70、PNPT1蛋白表达水平,采用流式细胞术检测缺氧+NC组、缺氧+Tom70过表达组细胞凋亡率,qPCR检测缺氧+NC组、缺氧+Tom70过表达组TUBA mRNA表达情况,免疫共沉淀技术检测细胞中Tom70与PNPT1的相互作用情况。结果(1)流式细胞术检测结果显示,与对照组比较,缺氧组细胞凋亡率明显增高(P<0.05)。qPCR检测结果显示,与对照组比较,缺氧组细胞中TUBA mRNA含量明显减少(P<0.05)。Western blotting检测结果显示,与对照组比较,缺氧组线粒体内PNPT1蛋白表达水平明显降低,而胞质PNPT1蛋白表达水平明显上升(P<0.05)。(2)Western blotting检测结果显示,与对照组和NC组比较,Tom70过表达组细胞内Tom70表达量明显增加(P<0.05);常氧条件下,NC组与Tom70过表达组线粒体及胞质中的PNPT1表达均无明显差异,而缺氧条件下,与缺氧+NC组比较,缺氧+Tom70过表达组线粒体PNPT1表达水平明显升高,胞质PNPT1表达水平明显降低(P<0.05)。免疫共沉淀结果显示,心肌细胞中的Tom70与PNPT1能够相互结合。流式细胞术检测结果显示,与缺氧+NC组比较,缺氧+Tom70过表达组细胞凋亡率下降(P<0.05)。qPCR检测结果显示,与缺氧+NC组相比,缺氧+Tom70过表达组TUBA mRNA表达水平明显增高(P<0.05)。结论Tom70可通过调控PNPT1的线粒体定位表达减少凋亡相关mRN
Objective To investigate the effect and mechanism of mitochondrial translocation regulation of translocase of outer mitochondrial membrane 70(Tom70)/polyribonucleotidyltransferase 1(PNPT1)in hypoxic injury of rat myocardial cells.Methods(1)Rat H9C2 cardiomyocytes were divided into control group(treated with normoxia for 12 hours)and hypoxia group(treated with hypoxia for 12 hours).The apoptosis rate of cardiomyocytes was detected by flow cytometry,the expression of TUBA mRNA was detected by qPCR,and the expression of PNPT1 protein was detected by Western blotting.(2)H9C2 cells were infected with lentivirus vector carrying Tom70 sequence to up-regulate the expression of Tom70.The cells were then divided into NC group(lentivirus negative control group,containing lentivirus empty vector only),Tom70 over-expression group(lentivirus vector carrying Tom70 sequence),hypoxia+NC group,hypoxia+Tom70 over-expression group.Western blotting was used to detect the expression level of Tom70,PNPT1 protein in cells,and flow cytometry was used to detect the apoptosis rate of hypoxia+NC group and hypoxia+Tom70 over-expression group,the expression of TUBA mRNA in the hypoxia+NC group and hypoxia+Tom70 overexpression group were detected by qPCR,and the interaction between Tom70 and PNPT1 in the cells was detected by immunoprecipitation technique.Results(1)The results of flow cytometry showed that compared with control group,the apoptosis rate of myocardial cells in hypoxic group was significantly higher(P<0.05).The results of qPCR showed that compared with the control group,the content of TUBA mRNA in cells of hypoxia group was significantly decreased(P<0.05).Western blotting showed that compared with control group,the expression level of PNPT1 protein in mitochondria of hypoxic group was significantly decreased,while the expression level of PNPT1 protein in cytoplasm was significantly increased(P<0.05).(2)Western blotting showed that compared with control group and NC group,the expression of Tom70 in the Tom70 overexpression group w
作者
程旭
田静
李静
刘青
裴海峰
Cheng Xu;Tian Jing;Li Jing;Liu Qing;Pei Hai-Feng(Medical College,Southwest Jiaotong University,Chengdu,Sichuan 610031,China;Department of Cardiovascular Medicine,Western Theater General Hospital,Chengdu,Sichuan 610083,China;Department of Medical Engineering,the 950th Hospital of Chinese PLA,Kashi,Xinjiang 844999,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2023年第6期663-669,共7页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金面上项目(81970241)
西部战区总医院院管重点项目(2021-XZYG-A03)。
关键词
心肌细胞凋亡
缺氧
多核糖核苷酸核苷转移酶1
线粒体外膜转运蛋白70
myocardial cell apoptosis
hypoxia
polyribonucleotide nucleoside transferase 1
translocase of outer mitochondrial membrane 70
