体外循环心内直视手术患者心肌细胞凋亡基因表达的变化  

Changes in expression of apoptosis genes in myocardial cells of patients undergoing open heart surgery with cardiopulmonary bypass

作　　者：王焰斌[1] 杨旭东[3] 李刚[2] 刘志红[2] 王湘[2] 郭建洲[2] 于洪涛[2] 张锐[2] 杨建安[2] 黄志勇[1] 

机构地区：[1]广东省深圳市孙逸仙心血管医院麻醉科,518020 [2]广东省深圳市孙逸仙心血管医院心外科,518020 [3]西安交通大学医学院生物化学和分子生物学系

出　　处：《中华麻醉学杂志》2015年第8期913-918,共6页Chinese Journal of Anesthesiology

基　　金：2013年广东省医学科学基金（A2013586）

摘　　要：目的 探讨体外循环心内直视手术患者心肌细胞凋亡基因表达的变化.方法 体外循环下行心脏瓣膜置换术的患者30例,性别不限,年龄21～59岁,心功能Ⅱ或Ⅲ级,ASA分级Ⅱ或Ⅲ级.于体外循环前3 min和主动脉开放1h时随机取15例患者,取右心耳组织,采用TUNEL法检测心肌细胞凋亡指数（AI）,采用人类全基因组表达谱芯片分析凋亡基因的差异表达情况.采用实时定量PCR法测定凋亡诱导因子（AIF）、凋亡蛋白活化因子1（APAF1）、细胞色素c（Cyt c）、Bax、Bc1-2、caspase-3、caspase-9、caspase-6、caspase-7的mRNA表达水平,从而对差异基因的注释系统（GO）及信号通路分析进行mRNA水平验证,采用Western blot法测定心肌组织Cyt c、Bax、Bcl-2和caspase-9的蛋白表达水平,从而对信号通路分析进行蛋白水平验证.结果 与体外循环前比较,主动脉开放1h时差异基因有1 615个,其中表达上调基因数为903个,表达下调基因数为712个.差异基因GO分析显示,17个基因与线粒体凋亡通路相关,其中14个基因上调,3个基因下调.差异基因信号通路分析显示,可信值高的前10位中,缺血再灌注诱导的线粒体凋亡信号通路位于首位.与体外循环前比较,主动脉开放1 h时患者心肌细胞AI升高,心肌组织AIF、APAF1、Cyt c、caspase-9、caspase-3、caspase-6、caspase-7和Bax的mRNA表达明显上调,Bcl-2 mRNA表达明显下调,心肌Cyt c、caspase-9和Bax蛋白表达明显上调,Bcl-2蛋白表达明显下调（P＜0.01）.该结果与基因表达谱芯片GO分析和信号通路分析结果一致.结论 线粒体凋亡通路是体外循环心内直视手术患者心肌细胞凋亡过程中的优势表达途径.Objective To investigate the changes in the expression of apoptosis genes in the myocardial cells of patients undergoing open heart surgery with cardiopulmonary bypass （CPB）.Methods Thirty NYHA class Ⅱ or Ⅲ and ASA physical status Ⅱ or m patients of both sexes, aged 21-59 yr, scheduled for elective open heart surgery with CPB, were enrolled in the study.At 3 min before CPB and 1 h after aortic unclamping, 15 patients were selected randomly, and myocardial specimens were obtained from the right atrium for detection of apoptosis （by TUNEL） and the differentially expressed apoptosis genes was analyzed using NimbleGen Human Gene Expression Microarrays.The apoptotic index was calculated.The expression of apoptosis-inducing factor （AIF） , apoptotic protease activating factor-1 gene （APAF1）, cytochrome c （Cyt c）, Bax, Bcl-2, caspase-3, caspase-9, caspase-6, and caspase-7 mRNA was detected by using quantitative real-time reverse transcriptase-polymerase chain reaction.Gene Ontology （GO） and pathway analyses of the differentially expressed genes were performed.The expression of Cyt c, Bax, Bcl-2 and caspase-9 was detected by Western blot, and pathway analysis of the differentially expressed genes was performed.Results Compared with that before CPB, there were 1 615 differentially expressed genes, and among the 1 615 genes, the number of up-regulated genes was 903, and the number of down-regulated genes was 712 at 1 h after aortic unclamping.GO analysis of the differentially expressed genes showed that 17 genes were closely related to the mitochondrial apoptotic pathway, and among the 17 genes, 14 genes were significantly up-regulated, and 3 genes were downregulated.The pathway analysis of the differentially expressed genes showed that of the top 10 that could be considered much more reliable, ischemia-reperfusion-induced mitochondrial apoptotic pathway was in the first place.Compared with that before CPB, apoptotic index was significantly increased, the expression of AIF, APAF1, Cyt c, caspas

关 键 词：心肌再灌注损伤 心肺转流术 细胞凋亡 微阵列分析 

分 类 号：R614[医药卫生—麻醉学]