电刺激迷走神经对大鼠缺血再灌注心肌基因表达影响的实验研究

The experimental study of effect of electric vagal stimulation on the gene expression of injured myocardium from ischemia/reperfusion rat model

目的 研究电刺激迷走神经对大鼠缺血再灌注心肌基因表达的影响以从基因水平探讨心肌保护机制.方法 将20只SD雄性大鼠按随机数字表法分成2组,缺血再灌注组（I/R组）和迷走神经电刺激组（STM组）,每组10只.通过结扎冠状动脉左前降支30 min后,松开缝线使冠状动脉再通120 min的方法制备心肌缺血再灌注模型.STM组在缺血再灌注前10 min开始以5V、2 ms、1 Hz强度持续电刺激颈部左侧迷走神经20 Bin.再灌注120 min后将每组按随机数字表法分为2部份,一部份取左心室心肌组织测定心肌梗死面积;一部分取左心室缺血区心肌组织成功提取RNA后,获得心肌基因表达谱.运用GeneSpring软件筛选出两组间差异表达显著的基因并进行分析,对其中具有代表意义的基因进行实时定量逆转录多聚酶链反应（Real-time PCR）验证.结果 （1）STM组心肌梗死区面积较I/R组明显缩小（P＜0.05）.（2）基因表达谱筛选得到186个差异表达显著的基因或探针,基因本体分析（GO）显示主要影响了3类基因的改变,上调代表基因与心肌保护有关;下调代表基因与炎症有关.（3）Real-time PCR结果和基因芯片表达的结果相符.结论 电刺激迷走神经对缺血再灌注心肌具有保护作用,其心肌基因表达谱发生了很大的变化,可能通过兴奋胆碱能抗炎通路来减轻和抑制心肌缺血再灌注损伤.

Objectives To investigate the effect of electric vagal stimulation on the gene expression of injured myocardium from ischemia/reperfusion rat model and explore the involved molecular mechanism.Methods Twenty Sprague Dawley male rats were randomly selected and divided into 2 groups evenly：ischemia/reperfusion group （I/R group）,and vagus nerve stimulation group （STM group）.The left anterior descending coronary artery （LAD） was ligated and subjected to 30 min of myocardial ischemia followed by 2 h of reperfusion.In addition,10 min before reperfusion,left cervical vagus nerve of STM group was subjected to electronic stimulation at 5 V,2 ms and 1 Hz for 20 min.After 120 min of reperfusion,every group was randomly divided into two parts.One part that myocardium was collected from left ventricle was applied to determine the area of myocardial infarction.The RNA isolated from another part of the ischemic myocardium collected from left ventricle was hybridized to get gene expression profiles and the quality of hybridized RNA from both I/R and STM group was assessed and analyzed.GeneSpring software was applied to screen out the genes,which show significant difference between groups I/R and STM.Real-time polymerase chain reaction （RT-PCR） was applied to analyze the expression of important genes.Results （1）The area of myocardial infarction of STM （25.5 ± 3.9） ％ was significant reduced relative to L/R group （45.5 ± 4.8） ％ （P ＜ 0.05）.（2）The expression levels of 186 genes were changed significantly,and analyzed by Gene ontology （GO） software,there were 3 kinds of genes were affected.The upregulated genes were reported to show protective effect on myocardium.The downregulated gene was relative to inflammation.（3）The RT-PCR result confirmed the genechip assay.Conclusions Electric vagal stimulation can reduce myocardial I/R injury in rats.Significant change of the gene expression was detected between groups I/R and STM.The results suggest that activation of cholinergic anti-inflammatory pathway be involved in the mechanism.