筛选心肌缺血再灌注大鼠差异表达基因

Screening of differentially expressed genes following myocardial ischemia/reperfusion in rats

目的:利用抑制性消减杂交技术,筛选出大鼠心肌缺血再灌注的差异表达基因,以期通过基因线索探讨其损伤机制。方法:实验于2006-03/10在中山大学中山医学院完成。①实验分组:Sprague-Dawley雄性大鼠40只,随机分为手术组,对照组,每组20只。②实验干预:手术组结扎左冠状动脉,心电图出现急性心肌梗死改变90min后,去除结扎线,再灌注60min,建立心肌缺血再灌注大鼠模型。对照组仅穿线不结扎,余同手术组。③取缺血区心肌,提取TotalRNA,构建cDNA文库,利用抑制性消减杂交技术筛选差异表达基因,测序,登录Genbank寻找同源性基因。结果:共获得124个阳性结果,56个为高表达基因,68个为低表达基因,其中发现5个新的cDNA片段。其中能量代谢、物质运输、信号转导相关差异表达基因分别占所有差异表达基因的39.25%,15.89%,15.89%,并且主要变化为下调。结论:抑制性消减杂交技术是一种高效的筛选差异基因的方法。心肌缺血再灌注后基因变化涉及多种功能的基因,以能量代谢、物质运输、信号转导相关基因下调明显。

AIM： Using suppression subtractive hybridization （SSH）, this study aims to investigate genetic clues and reveal the mechanisms of rat heart injuries caused by myocardial ischemia/reperfusion. METHODS. The experiment was carded out in the Zhongshan School of Medicine, Sun Yat-sen University from March to October in 2006. （1）Forty Sprague-Dawley male rats were randomized into operation group and control group, each contained 20 mice. （2）Myocardial ischemia/reperfusion rat models were established by left coronary artery ligation, followed by 90-minute acute myocardial infraction and 60-minute reperfusion. Control group was only sutured. （3）Total RNA were extracted from ischemic myocardium in rat models. After the synthesis of the cDNA, differentially expressed genes were isolated with SSH. Then the isolated genes were cloned and sequenced. The sequencing results were compared with their counterparts in Genbank to search for their homology. RESULTS： Among the 124 obtained positive genes, 56 genes were highly expressed and 68 lowly. Additionally, 5 new sequences of cDNA fragment were found. Energy metabolism, transport and signal transduction genes were 39.25%, 15.89%, 15.89%, respectively. And they all presented a decreasing tendency. CONCLUSION： SSH is an effective method to isolate differentially expressed genes. Myocardial ischemia/reperfusion changes the expressions of some genes in rat heart, such as the decrease of energy metabolism, transport and signal transduction associated genes.