AKAP1过表达腺病毒载体构建及其在心肌细胞中的功能初步研究  

Preliminary study on functions of AKAP1 in mouse primary cardiomyocytes

作　　者：刘峰舟 尹纯[2] 孙夏承 杨芳[2] 季乐乐[2] 李飞[1] 吴有盛[2] 

机构地区：[1]第四军医大学西京医院心血管内科,陕西西安710032 [2]第四军医大学基础部教学实验中心,陕西西安710032

出　　处：《心脏杂志》2017年第4期405-410,共6页Chinese Heart Journal

基　　金：国家自然科学基金项目资助(81400197;81170183);陕西省社会发展攻关项目资助(2013SF2-02)

摘　　要：目的利用Ad Easy腺病毒载体系统构建A激酶锚定蛋白(AKAP)1重组腺病毒载体,初步探索AKAP1在心肌细胞中的功能。方法以正常小鼠心肌细胞c DNA为模板,利用PCR获取AKAP1基因,构建腺病毒重组质粒p Ad Easy-AKAP1。用293A细胞,包装重组腺病毒Ad-AKAP1颗粒,并用荧光蛋白标记法测定其滴度。用Western blot检测重组腺病毒感染原代小鼠心肌细胞和糖尿病心肌病细胞模型后AKAP1、Caspase-3的表达情况,用流式细胞仪检测原代小鼠心肌细胞内ROS生成水平及AKAP1对高糖状态下心肌细胞凋亡的影响。结果构建了携带AKAP1基因的过表达腺病毒载体,并获得5×10^(10)pfu/ml的高滴度重组腺病毒。重组腺病毒可感染原代心肌细胞和糖尿病心肌病模型H9c2细胞并介导AKAP1过表达(P<0.05)。与阴性对照组相比,过表达AKAP1可抑制高糖高脂状态下心肌细胞内ROS的生成(P<0.05),可抑制高糖状态下心肌细胞的凋亡和Caspase-3活化(P<0.01)。结论过表达AKAP1对高糖高脂状态下的心肌细胞具有潜在保护作用,对心肌功能发挥具有重要作用。其机制可能与AKAP1抑制心肌细胞内ROS生成、抑制Caspase-3活化、抑制细胞凋亡有关。AIM To investigate the functions of A kinase anchoring protein 1 （ AKAP1 ） in cardiomyo- eytes by construction of AKAP1 recombinant adenovirus vector using AdEasy adenovirus vector system. METHODS AKAP1 was PCR-amplified using murine cDNA as a template and then recombinant Ad plasmid pAdEasy-AKAP1 was constructed. The recombinant adenovirus Ad-AKAP1 was packaged and amplified by using 293A cells. The virus titer was determined by green fluorescent protein labeling method. AKAP1 and Caspase-3 expression levels in mouse primary cardiomyocytes and diabetic cardiomyopathy model cells infecting recombinant adenovirus were detected by Western blot. The levels of ROS in mouse primary cardiomyocytes and the effect of AKAP1 on apoptosis of diabetic cardiomyopathy cells exposed to high glucose were detected by flow cytometry. RESULTS The recombinant adenovirus carrying AKAP1 gene was constructed and high titer recombinant adenovirus of 5 x 10^10 pfu/ml was obtained. The recom- binant adenovirus can infect primary myocardial ceils and diabetic cardiomvooathv model H9e2 cells andmediates AKAP1 overexpression （P 〈 0. 05）. Compared with the negative control group, overexpression of AKAP1 could inhibit the formation of ROS in cardiomyocytes （P 〈 0. 05 ） and of cardiac myocytes and the activation of Caspase-3 （ P 〈 0. 01 ） under high could inhibit the apoptosis glucose and fat condition. CONCLUSION Overexpression of AKAP1 has a potential protective effect on cardiomyocytes under high glucose and fat condition, which plays an important role in myocardial function. The mechanism may be related to which AKAP1 inhibited the formation of ROS, inhibited the activation of Caspase-3 and inhibition the apoptosis in cardiomyocytes.

关 键 词：A激酶锚定蛋白1 心肌功能 活性氧簇 腺病毒载体构建 

分 类 号：Q343.1[生物学—遗传学]