【作者】 陈晓凤；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2009， 博士

【摘要】 目的:研究胆碱能抗炎通路对大鼠缺血再灌注心肌基因表达的影响,并探讨其可能的分子机制。方法:（1）选择20只SD雄性大鼠随机分成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。分离颈部左侧迷走神经,暴露胸腔,在动脉圆锥与左心耳根部间结扎冠状动脉左前降支（left anterior descending coronary artery,LAD）,结扎30min后,松开缝线使冠状动脉再通120min。STM组在缺血再灌注前10min开始以5V、2ms、1HZ强度持续电刺激颈部左侧迷走神经20min。（2）缺血前由尾静脉及再灌注120min后由右心房各取0.5ml血测血浆肌钙蛋白I（Cardiac Troponin I,cTn-I）。再灌注120min后将每组随机分为2部份,一部份（n=7）取左心室心肌组织采用Evan’s Blue和TTC双染色法测定心肌梗死面积;一部分（n=3）取左心室缺血区心肌组织成功提取RNA后,与Affymetrix大鼠U230 2.0芯片杂交,获得I/R组与STM组心肌基因表达谱。（3）运用GeneSpring软件筛选出两组间差异表达显著的基因,对其进行层级聚类分析,基因本体分析（Gene ontology,GO）及信号传导通路（Pathway）分析。根据分析结果及相关文献查阅,探讨胆碱能抗炎通路对缺血再灌注心肌基因表达影响可能的分子机制,并对其中5个具有代表意义的基因（Bdkrb2,Ntrk1,Cd40lg,ATP2a2,Ctnnb1）进行实时定量逆转录多聚酶链反应（Real-Time PCR）验证。结果:（1）cTn-I表达两组缺血前无差异（I/R组:4.52±0.56ng/ml,STM组:4.50±0.57ng/ml,p>0.05）;STM组在再灌注120min时点（32.33±6.25ng/ml）明显小于I/R组（64.35±5.89ng/ml）（P<0.05）。（2）各组间心肌缺血区范围无明显差异（I/R组:48.2±4.5%,STM组:47.5±4.6%,p>0.05）。STM组心肌梗死区面积（25.5±3.9%）较I/R组（45.5±4.8%）明显缩小（P<0.05）。（3）基因表达谱筛选得到186个差异表达显著的基因或探针,其中上调基因或探针为133个,下调基因或探针为53个。GO分析显示主要影响了3类基因的改变:第一类主要涉及膜表面受体,代表基因为:Bdkrb2,Ntrk1,Cd40lg;第二类主要为ATP酶,ATP结合蛋白以及Ca离子以及其他离子转运蛋白,代表基因为:ATP2a2;第三类主要为转录调控因子,代表基因为Ctnnb1。其中上调基因:Bdkrb2,ATP2a2,Ntrk1,Ctnnb1文献报道与心肌保护有关;下调基因Cd40lg与炎症有关。Pathway分析显示差异基因参与30条信号通路,包括Wnt通路。（4）Real-time PCR结果和基因芯片表达的结果相符。STM组中,Bdkrb2,ATP2a2,Ntrk1,Ctnnb1均上调,Cd40lg下调。结论:胆碱能抗炎通路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,主要涉及膜表面受体,ATP酶、ATP结合蛋白以及Ca离子以及其他离子转运蛋白,转录调控因子等。提示胆碱能抗炎通路可能在抗炎的同时能通过兴奋心肌M₂受体使缓激肽受体B2（Bdkrb2）表达上调,神经生长因子受体（Ntrk1）表达上调,Cd40lg表达下调,肌浆网Ca²⁺泵（ATP2a2）表达上调,Wnt/β-catenin（Ctnnb1）信号通路的激活等几方面来减轻和抑制心肌缺血再灌注损伤。更多还原

【Abstract】 Objectives:to study the effect of cholinergic anti-inflammatory pathway on the gene expression of injured myocardium from ischemia/reperfusion rat model and investigate the involved molecular mechanism.Methods:(1) 20 Sprague Dawley male rats were randomly selected and divided into 2 groups evenly,the ischemia/reperfusion group(I/R group) and the vagus nerve stimulation group(STM group).The left cervical vagus nerve was isolated and the chest was exposed.The left anterior descending coronary artery(LAD) was ligated at the position between arterial cone and the root of the left atria appendage.Each group was subjected to 30 min of myocardial ischemia followed by 2h of reperfusion.In addition,10 min before reperfusion,left cervical vagus nerve of STM group was subjected electronic stimulation at 5V、2ms and 1HZ for 20min.(2) 0.5 ml blood was taken from tail vein before ligation and from right atrium after 120 min reperfusion for plasma troponin cTn-I analysis. After 120 min of reperfusion and 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 by Evan’s blue dye perfusion and triphenyl tetrazolium chloride(TTC) staining.The RNA isolated from another part of the ischemic myocardium collected from left ventricle was hybridized to Affymetrix GeneChip Rat Genome U230 2.0 and the quality of hybridized RNA from both I/R and STM group was assessed and analyzed.(3)GeneSpring software was applied to screen out the genes,which show significant difference between I/R group and STM group.Data was assessed by hierarchical clustering analysis and gene ontology(GO) analysis.The involved signaling pathway was investigated as well.Based on the results and relative reported papers,the molecular mechanism of the effect of cholinergic anti-inflammatory pathway on gene expression in ischemia/ reperfusion myocardium was investigated.Real-time PCR was applied to analyze the expression of following important genes:Bdkrb2, Ntrk1,Cd401g,ATP2a2,and Ctnnb1.Results:(1) There is no significant difference of cTn-1 level between I/R and STM group before the ligation.(I/R group:4.52±0.56 ng/ml,STM group:4.50±0.57 ng/ml,p＞0.05) After 120 min of reperfusion,the level of cTn-1 in STM group(32.33±6.25 ng/ml) was significant lower than I/R group(64.35±5.89ng/ml)(P＜0.05).(2)There was no significant difference of the area of myocardial ischemia between two groups(I/R:48.2±4.5%,STM:47.5±4.6%,p＞0.05). However,the area of myocardial infarction of STM(25.5±3.9%) was significant reduced compared with I/R group(45.5±4.8%)(p＜0.05).(3) The expression level of 186 genes were changed significantly,in which 133 genes were upregulated and 53 genes were downregulated. Analyzed by GO software,there were 3 kinds of genes were affected.The first kind is membrane receptors,such as Bdkrb2,Ntrk1 and Cd401g.The second is ATPase,ATP binding protein and calcium binding protein,for example ATP2a2.The last is transcriptional factors,such as Ctnnb1.The upregulated genes,which are Bdkrb2,ATP2a2,Ntrk1 and Ctnnb1,were reported to show protective effect on myocardium.The downregulated gene,Cd401g,was relative to inflammation.These genes involved in 40 signaling pathways,including Wnt pathway.(4) The result of real-time PCR confirmed the genechip assay,which showed the level of Bdkrb2,ATP2a2,Ntrk1 and Ctnnb1 were upregulated and Cd401g was downregulated.Conclusion:The activation of cholinergic anti-inflammatory pathway shows the protective effect on the ischemia/ reperfusion myocardia.Significant change of the gene expression was detected between I/R and STM group,such as membrane receptors,ATPase,ATP binding protein and calcium binding protein and transcriptional factors. The results suggest that activation of cholinergic anti-inflammatory pathway upregulated the Bdkrb2,Ntrk1 and ATP2a2,downregulated Cd401g and activated Wnt/β-catenin(Ctnnb 1) pathway.The sum of the evidences suggests that activation of cholinergic anti-inflammatory pathway relieve and prevent the myocardia from ischemia/ reperfusion injury.更多还原