肿瘤坏死因子受体相关蛋白1对大鼠缺氧心肌细胞保护作用的机制  

Mechanism of protective effects of tumor necrosis factor receptor associated protein 1 on hypoxic cardiomyocytes of rats

作　　者：向飞[1] 张东霞[1] 马思远[1] 黄跃生[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所 创伤、烧伤与复合伤国家重点实验室,重庆400038

出　　处：《中华烧伤杂志》2016年第12期744-751,共8页Chinese Journal of Burns

基　　金：国家自然科学基金（81101426、81571898）

摘　　要：目的探讨肿瘤坏死因子受体相关蛋白1（TRAP1）对大鼠缺氧心肌细胞保护作用的机制。方法取1～3d龄SD大鼠乳鼠，分离培养心肌细胞，用于以下实验。（1）取细胞，按随机数字表法（分组方法下同）分为TRAP1组和对照组，提取细胞总蛋白。TRAP1组细胞总蛋白加入小鼠抗大鼠TRAP1单克隆一抗，对照组细胞总蛋白加入小鼠来源的同型IgG，免疫共沉淀法和蛋白质谱分析检测TRAP1可能作用的蛋白。（2）取细胞，分为常氧空白对照组、常氧＋TRAP1干扰对照组、常氧＋TRAP1干扰组、常氧＋TRAP1过表达对照组、常氧＋TRAP1过表达组，每组1孔。常氧空白对照组细胞常规培养，后4组细胞分别加入TRAP1RNA干扰空病毒载体、TRAP1RNA干扰腺病毒载体、TRAP1过表达空病毒载体、TRAP1过表达腺病毒载体。另取细胞，分为常氧空白对照组、缺氧空白对照组、缺氧＋TRAP1干扰对照组、缺氧＋TRAP1干扰组、缺氧＋TRAP1过表达对照组、缺氧＋TRAP1过表达组，每组1孔。各缺氧组细胞同前对应的常氧组细胞处理后，缺氧6h。实时荧光定量RT—PCR检测各组细胞中细胞色素C氧化酶亚基Ⅱ（COXⅡ）mRNA表达。本实验重复3次。（3）取细胞，分为常氧空白对照组、缺氧空白对照组、缺氧＋TRAP1过表达对照组、缺氧＋TRAP1过表达组、缺氧＋TRAP1过表达＋COXⅡ干扰对照组、缺氧＋TRAP1过表达＋COXⅡ干扰组，每组3孔。前4组细胞的处理同（2），后2组细胞分别加入COXⅡRNA干扰空病毒载体、COXⅡRNA干扰腺病毒载体转染后，再分别加入TRAP1过表达腺病毒载体。细胞计数试剂盒8及酶标仪检测细胞增殖活性，碘化丙啶和Hoechst33342染色检测细胞死亡率。另取细胞，分为常氧空白对照组、缺氧空白对照组、缺氧＋TRAP1干扰对照组、缺氧＋TRAP1干扰组、缺氧＋TRAP1干扰＋COXⅡ过表达对照组、缺氧＋TRAP1干扰＋COXⅡ过表达�Objective To investigate the mechanism of protective effects of tumor necrosis factor receptor associated protein 1 （TRAP1） on hypoxic cardiomyocytes of rats. Methods Primary cultured cardiomyoeytes were obtained from neonatal Sprague-Dawley rats （aged 1 to 3 days） and then used in the following experiments. （1） Cells were divided into group TRAP1 and control group aeeording to the random number table （the same grouping method below） , and then the total protein of cells was extracted. Total protein of cells in group TRAPI was added with mouse anti-rat TRAP1 monoclonal antibody, while that in control group was added with the same type of IgG from mouse. Co-immunoprecipitation and protein mass speetrography analysis were used to determine the possible proteins interacted with TRAP1. （2） Cells were divided into normoxia blank control group （NBC） , normoxia ＋ TRAP1 interference control group （NTIC） , normoxia ＋TRAP1 interference group （NTI）, normoxia ＋ TRAP1 over-expression control group （NTOC） , and normoxia ＋ TRAP1 over-expression group （NTO）, with 1 well in each group. Cells in group NBC were routinely cultured, while cells in the latter four groups were respectively added with TRAP1 RNA interference empty virus vector, TRAP1 RNA interference adenovirus vector, TRAP1 over-expression empty virus vector, and TRAP1 over-expression adenovirus vector. Another batch of cells were divided into group NBC, hypoxie blank control group （HBC） , hypoxie ＋ TRAP1 interference control group （HTIC） , hypoxic ＋ TRAP1 interference group （HTI） , hypoxie ＋ TRAP1 over-expression control group （HTOC） , and hypoxic ＋ TRAP1 over-expression group （HTO） , with 1 well in each group. Cells in hypoxic groups were under hypoxic condition for 6 hours after being treated as those in the corresponding normoxia groups, respectively. The mRNA expression of cytoehrome e oxidase subunit Ⅱ （ COX Ⅱ ） of cells in each group was detected by real time fluorescent quantitiv

关 键 词：肌细胞 心脏 缺氧 细胞增殖 细胞死亡 肿瘤坏死因子受体相关蛋白1 细胞色素c氧化酶亚基Ⅱ 

分 类 号：R644[医药卫生—外科学]