微小RNA-21对缺血缺氧大鼠心肌细胞凋亡的影响  

Effects of microRNA-21 on the myocardial cell apoptosis induced by ischemia and hypoxia in rat

作　　者：谢琼慧[1] 赵超莉[1] 叶子青[1] 杨飞[1] 阮琼芳[1] 谢卫国[1] 

机构地区：[1]武汉大学同仁医院暨武汉市第三医院烧伤研究所,430060

出　　处：《中华烧伤杂志》2014年第2期153-157,共5页Chinese Journal of Burns

基　　金：武汉市卫生局科研项目（WX13A08）;武汉市科技局重点攻关项目（20056007071）

摘　　要：目的探讨微小RNA．21对缺血缺氧所致大鼠心肌细胞凋产的影响，并分析其可能的作用机制。方法（I）取大鼠心肌细胞株H9C2加入低糖无血清DMEM培养液模拟缺血环境，将细胞镫于气体成分为体积分数l％氧气、5％二氧化碳和94％氮气的i气培养箱中进行缺氧培养。实时荧光定量RT—PCR检测正常心肌细胞和缺血缺氧刺激6、24h心肌细胞中微小RNA-21的表达。（2）另取心肌细胞株H9C2按随机数字表法分为4组。正常对照组：不进行任何处理，常规培养；单纯缺咀缺氧组：行单纯缺血缺氧处理24h；阴性转染＋缺血缺氧组：转染微小RNA模拟物阴性对照24h后，给予缺血缺氧处理24h；微小RNA-2l＋缺血缺氧组：转染微小RNA-2l模拟物24h后，给予缺虹缺氧处理24h。后3组均于处理完毕后立即进行如下检测，正常对照组于同时相点收集细胞进行检测。流式细胞仪检测心肌细胞凋亡率；实时荧光定量RT-PCR和蛋白质印迹法分别测定心肌细胞程序性细胞死亡因子4（PDCD4）的mRNA和蛋白表达水平。本实验样本数为6或3。对数据行单因素方差分析和LSD-t检验。结果（1）正常心肌细胞和缺血缺氧刺激6、24h心肌细胞微小RNA-2l的相对表达量分别为1．09±0．17、0．75±0．08、0．67±0．08（F=11．280，P=0．009）。与正常心肌细胞比较，缺血缺氧处理24（t=4．461，P=0．004）、6h（t=3．642，P=0．011）的心肌细胞中微小RNA-21表达均下渊，且前者更明最。故后续实验细胞缺血缺氧处理均为24h。（2）正常对照组心肌细胞凋亡率为（3．5±0．7）％。缺血缺氧处理24h，单纯缺血缺氧组、阴性转染＋缺血缺氧组及微小RNA-21＋缺血缺氧组心肌细胞凋亡率分别为（17．3±3．2）％、（16．4±3．0）％、（7．6±2．0）％（F=15．176，P=0．001）。与正常对照组比较，单纯缺虹缺氧组心肌细胞凋亡率明显升高（t=5．64l，P�Objective To explore the effects of microRNA-21 on apoptosis of myocardial cell of rats as induced by ischemia and hypoxia, and to analyze the underlying mechanisms. Methods （ 1 ） Rat myo- cardial cell line H9C2 was cultured in a serum-free and low glucose DMEM medium using a hypoxic incuba- tor which was filled with 1% oxygen, 5% carbon dioxide, and 94% nitrogen to simulate ischemic environ- ment. The expression of microRNA-21 in normal myocardial cells and cells treated with low oxygen exposure for 6 and 24 h were assessed by real-time fluorescent quantitative RT-PCR. （2） Another portion of myocar- dial cells were divided into 4 groups according to the random number table ： normal control group （ NC, ordi- nary culture without any treatment） , ischemia/hypoxia group （IH, treated with ischemia and hypoxia for 24 h） , negative transfection control ＋ ischemia/hypoxia group （NC ＋ IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA mimics control for 24 h） , microRNA-21 ＋ ischemia/hypoxia group （ M ＋ IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA-21 mimics for 24 h）. The cells in the latter three groups were examined immediately after treatment, and cells in group NC were collected and examined at the same time point. Apoptosis rate of myocardial cells was determined by flow eytometer. The mRNA and protein expression levels of programmed cell death 4 （PDCD4） in myocardi- al cells were determined by real-time fluorescent quantitative RT-PCR and Western blotting respectively. The sample numbers in this experiment were 6 or 3. Data were processed with one-way analysis of variance and LSD- t test. Results （ 1 ） The expression level of microRNA-21 in normal myocardial cells and cells trea- ted with ischemia and hypoxia for 6 and 24 h were respectively 1.09 ± 0.17, 0.75 ± 0.08, and 0.67 ± 0.08 （ F = 11. 280, P = 0. 009）. Compared with expression level of microRNA-21 in normal myocardial cells, those of cells t

关 键 词：心肌缺血 细胞系 细胞凋亡 微小RNA-21 程序性细胞死亡因子4 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]