重组腺病毒介导反义p38α基因抑制烧伤复合缺氧条件下乳鼠心肌细胞凋亡的研究  

Study on adenovirus mediated antisense p38α fragment gene in suppressing apoptosis of the myocardium of neonatal rat by burn serum and hypoxia

作　　者：范鹏举[1] 沈艳 黄云 郑军[3] 黄晓元[1] 黄跃生[4] 

机构地区：[1]中南大学湘雅医院烧伤整形科,湖南长沙410008 [2]不详 [3]南华大学附属第一医院烧伤科 [4]第三军医大学西南医院烧伤研究所 创伤、烧伤、复合伤国家重点实验室

出　　处：《中国危重病急救医学》2010年第11期666-668,共3页Chinese Critical Care Medicine

基　　金：国家重点基础研究发展规划项目（G1999054202）

摘　　要：目的 探讨烧伤血清复合缺氧条件下乳鼠心肌细胞p38丝裂素活化蛋白激酶（p38MAPK）活化与细胞凋亡之间的关系.方法 采用原代培养的乳鼠心肌细胞,按照处理因素不同分为3组：正常对照组细胞自然生长;烧伤缺氧组细胞给予烧伤血清＋缺氧刺激（含10%的40%总体表面积Ⅲ度烫伤大鼠伤后6 h血清的DMEM/F12培养液＋1%O2、5%CO2和94%N2）;反义阻断组在细胞转染反义p38α重组腺病毒（AD-ASp38α）后给予烧伤血清＋缺氧刺激.细胞培养6 h后,采用蛋白质免疫印迹法（Western blotting）检测p38磷酸化水平;采用DNA片断和流式细胞术检测心肌细胞凋亡情况.结果 与正常对照组比较,烧伤缺氧组p38磷酸化水平（灰度值）明显提高（8.68±0.14比3.21±0.05,P＜0.01）,细胞凋亡率明显增高[（50.367±0.451）%比（2.063±0.111）%,P＜0.01];心肌细胞转染AD-ASp38α后,p38磷酸化水平（灰度值）明显下降（5.46±0.16比8.68±0.14,P＜0.01）,细胞凋亡率明显降低[（13.200±0.121）%比（50.367±0.451）%,P＜0.01].结论 烧伤血清复合缺氧条件下通过促使p38MAPK活化导致心肌细胞凋亡增多;阻断p38MAPK信号转导通路可减轻严重烧伤早期心肌细胞的损伤.Objective To investigate the relationship between the activation of p38 mitogen-activated protein kinase （p38MAPK） in the myocardium and the apoptosis in the presence of burn serum and hypoxia. Methods Ventricular myocardium isolated from neonatal rats were employed in this study, and they were divided into three groups as the normal control group, with the myocardium grew naturally; burn serum＋ hypoxia group, in which the myocardium was stimulated by the serum collected from the rat 6 hours after burn injury involving 40% of total body surface area （TBSA）, and at the same time exposed to 1%O2, 5% CO2, and 94 %N2; antisense blocking group, in which rats were pretreated by AD-antisense （AS） p38α, then exposed to the same conditions as burn serum＋hypoxia group.The phosphorylation of p38 in the myocardium was determined by Western blotting.The level of myocardium apoptosis was determined by DNA ladder and flow cytometry.Results Compared with normal control group, the level of phosphorylation of p38 （gray value） was markedly increased （8.68±0.14 vs.3.21±0.05, P＜0.01＝ after being exposed to burn serum and hypoxia, and at the same time myocardium apoptosis was strikingly increased[（50.367±0.451）% vs.（2.063±0.111）%, P＜0.01＝.When the myocardium was transfected by AD-ASp38α, the phosphorylation of p38 （gray level） was decreased remarkably （5.46±0.16 vs.8.68±0.14, P＜0.01＝, the rate of the apoptosis was lowered remarkably[（13.200 ± 0.121 ） % vs.（50.367 ± 0.451）%, P ＜ 0.01]. Conclusion Burn serum combined with hypoxia can induce apoptosis of the myocardium by activating p38MAPK;blockage of p38MAPK signal transduction pathway may lessen the damage of the myocardium in early period of severe burn.

关 键 词：P38丝裂素活化蛋白激酶 烧伤 缺氧 反义p38α重组腺病毒 心肌细胞 凋亡 

分 类 号：R686[医药卫生—骨科学]