pFLAG-AMPKα2真核表达质粒构建及对心肌缺氧/复氧损伤的作用

中国临床药理学与治疗学 ›› 2012, Vol. 17 ›› Issue (12): 1333-1338.

pFLAG-AMPKα2真核表达质粒构建及对心肌缺氧/复氧损伤的作用

刘丹¹, 钟斌³, 万青¹, 曾姝¹, 吴晓牧², 何明¹

¹南昌大学药学院药理学与分子治疗学教研室,南昌 330006,江西;
²江西省人民医院神经内科,南昌 330006,江西;
³赣南医学院第一附属医院, 赣州 341000,江西

收稿日期:2012-07-10 修回日期:2012-11-14 发布日期:2012-12-31
通讯作者: 何明,通信作者,男,博士,教授,博导,研究方向:心肌急性损伤与保护。Tel: 0791-86361839 E-mail: jxhm56@163.com;吴晓牧,通信作者,男,博士,教授,博导,研究方向:细胞内信号通路。Tel: 0791-86895722 E-mail: wuxm79@163.com
作者简介:刘丹,女,博士,副教授,研究方向:心肌急性损伤与保护。Tel: 0791-86362231 　E-mail: 385910780@qq.com
基金资助:
国家自然科学基金资助项目(81160402, 81072632, 81100104); 博士后科学基金资助(20110491496); 江西省自然科学基金资助项目(2010GQY0230)

Construction of eukaryotic plasmid of pFLAG-AMPKα2 and the effect on cardiomyocytes subjected to anoxia-reoxygenation injury

LIU Dan¹, ZHONG Bin³, WAN Qing¹, ZENG Shu¹, WU Xiao-mu², HE Ming¹

¹Nanchang University School of Medical College, Nanchang 330006, Jiangxi, China;
²Department of Neurology, Jiangxi Provincial People's Hospital, Nanchang 330006,Jiangxi, China;
³First Affiliated Hospital of Gannan Medical college, Ganzhou 341000, Jiangxi,China

Received:2012-07-10 Revised:2012-11-14 Published:2012-12-31

摘要/Abstract

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,Western 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1700 bp,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。

关键词: AMPKα2;, 缺氧/复氧损伤, 凋亡, ROS, 氧化应激

Abstract: 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 (P<0.01). 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.

Key words: AMPK, Anoxia/reoxygenation injury, Apoptosis, ROS, Oxidative stress

中图分类号:

R965.2

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.

LIU Dan, ZHONG Bin, WAN Qing, ZENG Shu, WU Xiao-mu, HE Ming. Construction of eukaryotic plasmid of pFLAG-AMPKα2 and the effect on cardiomyocytes subjected to anoxia-reoxygenation injury[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2012, 17(12): 1333-1338.

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