【作者】 李艳艳；

【导师】 熊光仲；

【作者基本信息】 中南大学 ， 急诊医学， 2008， 硕士

【摘要】 背景与目的:肢体缺血再灌注损伤临床上极为常见,外周血管手术、肢体的创伤、断肢再植术、血栓形成、腹主动脉瘤手术、挤压综合征等均可引起肢体缺血再灌注损伤。近年研究发现,肢体缺血再灌注损伤不仅影响局部缺血组织的存活和功能,而且还可造成全身炎症反应综合征,累及心、肝、肺、肾、小肠、脑等远隔器官、导致多器官功能受损,其中以心血管系统的损伤最为重要。目前对肢体缺血后所致的肺,脑,肝,肠损伤机制的研究较多,对骨胳肌缺血再灌注所致心肌损伤病理生理机制,至今尚未完全阐明,大量研究表明:严重的肢体缺血再灌注损伤可导致应激性的高血压,应激性心律失常,应激性心肌缺血,心功能不全等,血红素加氧酶-1(heme oxygenase-1,HO-1)是目前发现的受最多因素诱导的应激蛋白。这些刺激的共同点是能造成氧化应激。HO-1具有抗炎、抗细胞凋亡、抗增殖、抗氧化的性质,还有免疫应答等方面具有重要的作用,并成为近几年的研究热点。本实验应用止血带构建大鼠下肢缺血再灌注模型,观察肢体缺血再灌注后心肌病理学改变,氧化应激机制及诱导型血红素氧化酶(HO-1)的表达来说明心肌的损伤及自身保护机制。本实验分为两个部分:一,观察肢体缺血再灌注后心肌的损伤变化,并了解氧化应激在肢体缺血再灌注后继发的心肌损伤中的作用。二,观察内源性HO-1在肢体缺血再灌注后心肌中的表达变化的规律及其意义。方法:本实验应用止血带捆扎大鼠下肢来构造肢体缺血再灌注模型,将实验的大鼠随机分为七组。即:正常组,缺血4小时再灌注2小时组,再灌注4小时组,再灌注6小时组,再灌注8小时组,再灌注16小时组,再灌注24小时组。第一部分实验:应用光镜观察心肌组织的病理变化;分别测定血清及心肌组织中的丙二醛(MDA)含量、总超氧化物歧化酶(T-SOD)活力及髓过氧化物酶(MPO)活性。第二部分实验:应用反转录—多聚酶链反应(RT-PCR)检测心肌HO-1mRNA表达的变化规律。结果:缺血再灌注4,6小时,心肌细胞明显肿胀,肌间隙增宽,小血管充血明显,肌间隙内可见大量红细胞漏出,肌间隙有核细胞明显增多,再灌注24小时上述改变减轻。与正常组比较,血浆MDA及心肌MDA:缺血再灌注各组均明显升高(P＜0.01),再灌注4h达高峰;血浆SOD:缺血再灌注各组均明显下降(P＜0.01),再灌注4小时达最低值,心肌SOD:再灌注各组均明显下降(P＜0.01),再灌注8小时达最低值;血浆及心肌MPO:缺血再灌注2h～8h出现明显升高(P＜0.01),血浆MPO 4h达高峰,心肌MPO 6h达高峰,16,24h下降。肢体缺血再灌注可诱导HO-1mRNA在心肌表达上调,与正常组比较,再灌注2h,HO-1mRNA表达无显著变化(P＞0.05);再灌注4h,6h,8h,16h,24h后HO-1表达显著增强(P＜0.01),并在16h达高峰。结论:肢体缺血再灌注可造成心肌损伤,再灌注4-6小时心肌损伤最重,全身及心肌局部的炎细胞聚集活化,炎性因子的激活和过度的氧化应激及氧化抗氧化失衡是肢体缺血再灌注致心肌损伤的机制,机体存在自身保护机制,活性氧族及炎性因子能上调HO-1的表达,对抗氧化应激,从而发挥心肌细胞保护作用。更多还原

【Abstract】 Objective and background:ischemical reperfusion injury of limb is a common pathophysiological phenomenon clinically.Ischemia of the limbs is a unavoidable occurrence during peripheral vasculai surgery, insult of limbs、thrombogenesis、abdominal aneurysm surgery, replantation of extremities,peripheral vascular injury or crushing syndrome.Recent studies have shown that limb IR can trigger a systemic inflammatory response syndrome(SIRS).It causes not only local tissue injury,but also remote organs dysfunction including heart,liver,lung,kindy and small intestine.clinical studies suggest that the heart is at high risk in the setting of sirs.the mechanism of acute myocardial injury following limb IR is very complicated,up to now,not completely elucidated yet.a great quantity of study indicate that severe ischemical reperfusion injury of limb can induce irritable high blood pressure,arrhythmia,myocardial ischemia and cardia insufficiency,heme oxygenase-1 is a ubiquitous stress protein and is induced in many cell types by various stimuli.the this stimuli all can induce oxidative stress.The protective effects of HO-1 is very important via its antiinflammatory,antiapoptotic,antiproliferative, and antioxidant properties,as well as its effects on the immune response. In the present study,we have established the model of bilateral hindlimb ischemia and reperfusion in rats by using a tourniquet.To explain the mechanism of myocardial injury and autoprotection by observing pathologic change of myocardium,the mechanism of oxidative stress and the expression of HO-1mRNA..our experiment is divided into two parts:Firstly,we explore the role of oxidative stress in the myocardial injury following the limb ischemical reperfusion injury by observing change of myocardium.secondly,we are to ascertain the protective effects of endogenous HO-lon the myocardial injury during the limb ischemical reperfusion.Method:we have established the model of bilateral hindlimb ischemia and reperfusion in rats by using a tourniquet.the experimental rats were divided to 7 groups randomly.Igroup:Normal group;Ⅱgroup:a group of animals were subjected to suffer ischemic 4h and reperfusion 2h,Ⅲgroup:ischemic 4h and reperfusion 4h;Ⅳgroup:ischemic 4h and reperfusion 6h;Ⅴgroup:ischemic 4h and reperfusion 8h;Ⅵgroup: ischemic 4h and reperfusion 16h;Ⅶgroup:ischemic 4h and reperfusion24h.the first part:to observe pathological change of myocardium by using light microscope,to measure the content of MDA、T-SOD and MPO in the serum and myocardium.;the second part:to detect the expression of HO-1mRNA by using RT-PCR.Result:After reperfusion 4h,myocardial cells were engorged, Vascular engorgement and Small spatium intermusculares were widen obviously,a bulk of akaryocyte and nucleated cells were observed in the spatium intermusculare,above-mentioned change were lessen following reperfusion 24h.To compare with normal group normal,plasma and myocardium MDA:The levels of MDA,MPO in plasma and the levels of MDA,MPO in myocardium were significantly increased in all groups of IR(P＜0.01),and the myocardial and plasma levels of SOD were markedly decreased(P＜0.01).However,the plasma levels of MDA and MPO and the myocardial level of MDA achieved to high-peak after reperfusion for 4h,then they decreased,while,the myocardial level of MPO reach to high-peak following IR for 6h,the plasma level of SOD reach to nadir after IR for 4h,but the myocardial level of SOD reach to nadir following IR for 8h.limb IR can induce to up-regulation of HO-1mRNA,compared to grounp normal:the expression of HO-1 was not significant change after IR2h(P＞0.05).and it is markedly increased after IR4h,and up to peak after IR16h(P＜0.01).Conclusion:Limb IR can induce myocardial injury.the injury is the most serious during 4-6h after reperfusion.The mechanism may be involved in the activating of neutrophil and inflammatory cytokines,excess oxidative stress,the imbalance of oxidant-antioxidant system,organism have the mechanism of autoprotection,the up-regulation of endogenous HO-1 may play an important role in myocardial injury following limb IR.更多还原