中文摘要：

目的：构建大鼠pFLAG-AMPKα2真核表达质粒,分析AMPKα2过表达对心肌细胞缺氧/复氧损伤的影响。方法：提取H9c2心肌样细胞的mRNA,反转录为cDNA。PCR扩增AMPKα2基因的cDNA全长,并将其克隆至pFLAG-CMV-4构建pFLAG-AMPKα2重组质粒。转染pFLAG-AMPKα2至H9c2细胞,West-ern Blot测定AMPKα2的蛋白表达情况,建立缺氧/复氧（A/R）损伤模型,MTT法检测心肌细胞的存活率,生物自动分析仪检测LDH活性,流式细胞术检测各实验组心肌细胞凋亡程度、细胞内ROS的变化,试剂盒检测细胞内抗氧化酶（SOD、GSH-Px）活性。结果：AMPKα2全长基因序列克隆至真核表达载体pFLAG-CMV-4,酶切鉴定片段大小为1700bp,Western blot检测转染pFLAG-AMPKα2后,AMPKα2蛋白在H9c2细胞中高表达。H9c2细胞遭受A/R损伤,心肌细胞存活率下降,LDH活性增加,细胞凋亡加重,ROS生成增加,SOD及GSH-Px的酶活性下降,pFLAG-AMPKα2重组质粒的导入可明显逆转、改善上述各项指标,从而对抗A/R损伤。结论：构建成功的pFlAG-AMPKα2重组质粒能对抗A/R所致的心肌细胞损伤,其机制与改善心肌细胞的氧化应激作用有关。

英文摘要：

AIM： To construct eukaryotic plasmid pFLAG-AMPKα2,and explore the effect on cardiomyocytes subjected to anoxia/reoxygenation（A/R） injury.METHODS： Total mRNA was extracted from H9c2 cells,and cDNA was formed by reverse transcription.The AMPKα2 coding sequence was amplified by polymerase chain reaction（PCR） and cloned into pFLAG-CMV-4.The plasmid was transfected into H9c2 cells and AMPKα protein was detected by Western blot.A/R injury model was established,cell viability was detected by MTT assay,LDH activity was analyzed with an automatic biochemical analyzer.The percentage of apoptosis,and intracellular ROS were measured by flow cytometry.SOD and GSH-Px were measured by a colorimetric method.RESULTS：AMPKα2 was successfully constructed into pFLAG-CMV-4 the expression vector.The length of the fragment identified by restriction enzyme digestion was 1700 bp.AMPKα2 protein was significant increase after transfecting with pFLAG-AMPKα2.After anoxia/reoxygenation in H9c2 cells,LDH activity,ROS production,and apoptosis were remarkably increased,while cell viability,activities of SOD and GSH-Px were decreased compared with control group（P0.01）.However,the overexpression of AMPKα2 with transfecting pFLAG-AMPKα2 could reverse above index and then protect against A/R injury.CONCLUSION： Recombinant plasmid pFLAG-AMPKα2 could protect H9c2 cells against A/R injury and the mechanism involving improvement of oxidative stress.