中国临床药理学与治疗学2012，Vol.17Issue(12)：1333-1338,6.
pFLAG-AMPKα2真核表达质粒构建及对心肌缺氧/复氧损伤的作用
Construction of eukaryotic plasmid of pFLAG-AMPKα2 and the effect on cardiomyocytes subjected to anoxia-reoxygenation injury
摘要
Abstract
AIM: To construct eukaryotic plasmid pFLAG-AMPKa2, and explore the effect on cardiomyocytes subjected to anoxia/ reoxygenation (A/R) injury. METHODS: Total mRNA was extracted from H9c2 cells, and cD-NA was formed by reverse transcription. The AMPKα2 coding sequence was amplified by pol-ymerase chain reaction (PCR) and cloned into pFLAG-CMV-4. The plasmid was transfected into H9c2 cells and AMPKα protein was detec-ted by Western blot. A/R injury model was es-tablished, cell viability was detected by MTT assay, LDH activity was analyzed with an auto-matic 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 (P < 0. 01 ). However, the overexpression of AMPKα2 with transfecting pFLAG-AMPKα2 could reverse above index and then protect a-gainst A/R injury. CONCLUSION: Recombinant plasmid pFLAG-AMPKα2 could protect H9c2 cells against A/R injury and the mechanism in-volving improvement of oxidative stress.关键词
AMPKα2/缺氧/复氧损伤/凋亡/ROS/氧化应激Key words
AMPK/Anoxia/reoxygenation injury/Apoptosis/ROS/Oxidative stress分类
医药卫生引用本文复制引用
刘丹,钟斌,万青,曾姝,吴晓牧,何明..pFLAG-AMPKα2真核表达质粒构建及对心肌缺氧/复氧损伤的作用[J].中国临床药理学与治疗学,2012,17(12):1333-1338,6.基金项目
国家自然科学基金资助项目(81160402,81072632,81100104) （81160402,81072632,81100104）
博士后科学基金资助(20110491496) （20110491496）
江西省自然科学基金资助项目(2010GQY0230) （2010GQY0230）
