AMPKα2基因shRNA重组质粒的构建以及在氯离子介导的大鼠心肌缺氧/复氧损伤中的作用

 目的：构建AMP活化的蛋白激酶α2亚基(AMPKα2)基因shRNA重组表达质粒，转染H9c2心肌细胞，探讨其在氯离子(Cl^-)介导的心肌细胞缺氧/复氧（A/R）损伤中的作用。方法：构建靶向AMPKα2基因的shRNA重组质粒pSuper-AMPKα2 shRNA，转染H9c2细胞；Western blotting法测定AMPKα2的蛋白表达情况。实验分5组，每组重复6次：(1) Control组；(2) A/R组；(3) Cl^--free A/R组；(4) pSuper+Cl^--free A/R组；(5) pSuper-AMPKα2 shRNA+ Cl^--free A/R组。处理结束后，生化自动分析仪测定LDH活性，MTT法测定细胞存活率，流式细胞术检测细胞凋亡和活性氧（ROS）水平，试剂盒检测细胞内超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性。结果：重组表达质粒pSuper-AMPKα2 shRNA经测序证明构建正确；转染心肌细胞后，AMPKα2蛋白表达明显下降；Cl^--free A/R组较之A/R组细胞存活率升高，LDH含量下降，细胞凋亡减少，ROS生成减少，SOD和GSH-Px活性增加；转染pSuper-AMPKα2 shRNA质粒后，阻断了Cl--free液的保护作用，且ROS生成增加，SOD和GSH-Px活性也下降。结论：成功构建pSuper-AMPKα2 shRNA重组质粒。AMPKα2基因沉默阻断了Cl^--free液对心肌细胞A/R损伤的保护作用。

Effect of AMPKα2 gene silencing by shRNA recombinant plasmid on chloride-mediated anoxia/reoxygenation injury in rat cardiomyocytes

AIM: To explore the role of AMP-activated protein kinase α2 subunit (AMPKα2) gene in chloride-mediated anoxia/reoxygenation (A/R) injury by transfection of short-hairpin RNA (shRNA) expression vector targeting to AMPKα2 gene into H9c2 cardiomyocytes. METHODS: Recombinant shRNA expression vector pSuper-AMPKα2 targeting to AMPKα2 gene was constructed and transfected into H9c2 cardiomyocytes. The protein expression of AMPKα2 was determined by Western blotting. The cells were divided into 5 groups: control group, A/R group, Cl^--free A/R group, pSuper+Cl^--free A/R group and pSuper-AMPKα2 shRNA+Cl^--free A/R group. After treatment, the cell viability was detected by MTT assay. LDH activity was analyzed with an automatic biochemical analyzer. The apoptotic rate and the level of intracellular ROS was measured by flow cytometry. The activity of SOD and GSH-Px was analyzed by a colorimetric method. RESULTS: The result of sequencing proved that the recombinant plasmid pSuper-AMPKα2 shRNA was correctly constructed. The protein level of AMPKα2 significantly decreased after the plasmid was transfected into the cardiomyocytes. Compared with A/R group, the cell viability and the activity of SOD and GSH-Px were significantly increased, while the activity of LDH, apoptotic rate and ROS production were significantly decreased in Cl^--free A/R group. The protective effect of Cl^--free solution on the A/R-induced injury of cardiomyocytes was abolished, and the ROS production was increased and the activity of SOD and GSH-Px was decreased after the cells were transfected with pSuper-AMPKα2 shRNA. CONCLUSION: Recombinant plasmid pSuper-AMPKα2 shRNA is successfully constructed, and silencing of AMPKα2 gene abolishes the protective effect of Cl^--free solution on A/R injury.