【作者】 孙海梅；

【导师】 郭涛； 许汪斌； 申丽娟； 喻卓； 孟照辉；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 博士

【摘要】 背景与目的随着冠状动脉溶栓术、经皮冠状动脉介入治疗(PCI)和冠状动脉搭桥术(CABG)等在临床的广泛应用,急性心肌梗死进入了再灌注时代。虽然及时恢复冠脉灌注能挽救缺血心肌,但闭塞冠脉再通可进一步加重缺血心肌损伤,这种现象称缺血再灌注损伤(I/R)。深入研究心肌缺血再灌注损伤的病理生理机制及寻找防治心肌缺血再灌注损伤的措施是目前心血管领域研究的热点。作为一种启动机体内源性保护机制的方法,缺血预处理(IPC)对心肌缺血再灌注损伤的保护作用已经得到普遍认可。但由于缺血预处理需在长时间缺血前实施,限制了其在临床上的应用。近来,缺血后处理(Postcond)作为一种新的心肌保护措施引起了人们的广泛关注。缺血后处理是指在心肌长时间缺血后再灌注开始前实施的数个循环的短暂的血流灌注/阻断,能减轻随后的再灌注损伤。缺血后处理不需预知缺血时间,操作简便的特点使其具有广阔的临床应用前景。目前实验动物的研究表明,缺血后处理对小鼠、大鼠、家兔、犬等的心肌保护作用较为肯定,但对与人类心脏最接近的动物--猪的心肌保护作用却尚无定论;且目前采用闭胸式心梗模型研究缺血后处理心肌保护作用的研究尚不多见;同时对缺血后处理心肌保护作用的研究主要是从再灌注早期(3小时以内)抑制炎症反应、限制心梗面积及改善心功能等方面证实,而缺血后处理是否通过抑制细胞凋亡发挥长期的心肌保护作用尚不清楚。本研究选择猪作为研究对象,建立猪闭胸式心肌梗死模型,在此基础上观察缺血后处理对猪心肌的保护作用,探讨抗氧化作用及炎症介质在缺血后处理抗心肌缺血再灌注损伤中的作用,并通过观察比较缺血后处理对再灌注72小时的心肌生化标志物、心肌梗死面积、心肌超微结构改变、心肌细胞凋亡及细胞凋亡相关基因(Bcl-2,Bax)蛋白表达等指标的影响,探讨缺血后处理对心肌再灌注损伤晚期保护效应及作用机制,为临床心肌缺血再灌注损伤的防治提供新策略。方法1、猪闭胸式心肌梗死模型的建立健康滇南小耳猪被随机分为2组,(1)假手术组(Sham组):仅将球囊放至左前降支(LDA)远端,但不封堵冠状动脉;(2)缺血再灌注组(I/R组):球囊充气封堵LDA60min。监测心率和血压,行冠脉造影,心电图检测,检测心肌生化标志物包括磷酸肌酸激酶(CK)、乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)和肌钙蛋白(cTnI),并行心肌病理组织学检测。2、缺血后处理抗猪心肌缺血再灌注损伤的保护作用研究实验猪被随机分为4组,(1)假手术组(Sham组);(2)缺血再灌注组(I/R组);(3)缺血后处理组(Postcond组):在心肌缺血60min后即刻,通过球囊放气开通冠脉30s、球囊充气造成30s短暂再缺血,连续8次循环;(4)单纯缺血组(I组):将剪为1/2的球囊留置在LAD的远端,导致自体血栓形成,造成心肌缺血。检测心肌生化标志物包括CK、LDH、CK-MB及cTnI,心梗面积及心肌细胞的超微结构。3、缺血后处理对猪心肌缺血再灌注损伤的抗氧化和调控炎症介质作用的研究采用分光光度比色法分别检测血清丙二醛(MDA)及超氧化物歧化酶(SOD)活性的变化;采用ELISA法分别检测猪肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和白细胞介素10(IL-10)的浓度。4、缺血后处理对猪心肌缺血再灌注损伤的抗细胞凋亡的作用及其机制的研究采用电镜及TUNEL法检测猪心肌组织细胞凋亡细胞,并采用免疫组织化学法及Western blot法检测凋亡相关的调控基因(Bcl-2及Bax)蛋白表达的变化.结果1.猪闭胸式心肌梗死模型的成功建立球囊导管封堵LDA远端后,冠脉造影示远端无血流,出现典型的心电图改变如ST段抬高,T波高耸融合;再灌注后2h,24h,48h,72h时间点的CK、CK-MB、cTnI、LDH等心肌生化标志物指标均较缺血前明显增高;病理学检查符合心肌梗死的特点。通过冠脉造影、心电图、心肌生化标志物、病理学检查等方法确认猪闭胸式心肌梗死模型的成功建立。2.缺血后处理抗猪心肌缺血再灌注损伤的保护作用(1)心肌生化标志物:与I/R组比较,Postcond组再灌注后24h、48h及72h,CK-MB、cTnI、CK、LDH等指标在的明显降低(P<0.05),而I组在相应时间点的指标则明显升高(P<0.05)。(2)心肌梗塞面积的变化:Sham组未发生心肌梗死,I/R组、Postcond组及I组心肌梗塞的面积分别是23.26±3.13%,10.89±2.02%、33.3±12.2%。与I/R组相比,Postcond组的心梗面积明显降低(P<0.05)。(3)心肌细胞的超微结构的变化:缺血后处理可以有效地减轻心肌细胞超微结构的损伤。结果提示缺血后处理具有显著抗心肌缺血再灌注损伤的作用。3、缺血后处理对猪心肌缺血再灌注损伤的抗氧化和调控炎症介质作用Postcond组在再灌注2h和24h与I/R组相比,SOD活性明显增高,MDA水平明显下降(P<0.05);Postcond组在再灌注2h和24h与I/R组相比,TNF-α和IL-6浓度明显降低(P<0.05),IL-10浓度明显增高(P<0.05)。4、缺血后处理对猪心肌缺血再灌注损伤的抗细胞凋亡的作用及其机制的研究(1)猪的心肌细胞凋亡的变化:采用电镜和TUNEL法结果显示,Sham组没有发现凋亡阳性细胞,I组、I/R组、Postcond组均发现凋亡阳性细胞。与I/R组相比,Postcond组心肌梗死区、边缘区和非梗死区细胞凋亡指数(AI)明显降低(P<0.05),I组心肌梗死区、非梗死区AI较I/R组明显增加P<0.05)。(2)猪心肌细胞Bcl-2及Bax蛋白表达变化:Sham组心肌细胞有一定量的Bcl-2及Bax蛋白表达,I/R组心肌梗死区、边缘区和非梗死区的Bcl-2和Bax的蛋白表达及Bax/Bcl-2比值较Sham组增强(p<0.05)。与UR组相比,Postcond组能够明显地降低心肌梗死区、边缘区及非梗死区Bax的蛋白表达(p<0.05),明显增加边缘区Bcl-2蛋白表达(p<0.05),并可明显降低边缘区、非梗死区的Bax/Bcl-2的比值(p<0.05)。结论1、猪闭胸式心梗模型是研究缺血后处理的理想动物模型。2、缺血后处理可降低再灌注损伤72小时后心肌生化标志物的释放、限制心肌梗死面积、减轻心肌细胞超微结构损害,缺血后处理对猪缺血再灌注损伤晚期具有心肌保护作用。3、缺血后处理减轻脂质过氧化产物MDA,提高抗氧化物质SOD水平,提示后处理可能通过抗氧化作用发挥心肌保护作用。4、缺血后处理减少心肌缺血再灌注损伤后促炎细胞因子TNF-α、IL-6的产生,增加抗炎细胞因子IL-10的水平,可能通过调控炎性细胞因子平衡发挥心肌保护作用。5、缺血后处理抗猪心肌缺血再灌注损伤的作用可能与抗细胞凋亡的作用有关,可能是通过调控细胞凋亡相关基因Bax和Bcl-2蛋白的表达。6、本研究为缺血后处理临床应用提供理论和实验依据,为防治心肌缺血再灌注损伤的提供新的靶点,具有广阔的应用前景以及重要的应用价值和意义。更多还原

【Abstract】 Background and ObjectivesWith the application of thrombolytic therapy;percutaneous coronary intervention (PCI) and coronary artery bypass grafting(CABG) in the clinic,coronary reperfusion therapy has become established for the management of acute myocardial infarction. Although ischemic myocardium can be salvaged by timely reperfusion of the occluded artery,reperfusion may lead to detrimental consequences,which is called ischemia-reperfusion injury.The phenomenon is now known to involve extension of myocardial stunning,microvascular dysfunction and other damages over reperfusion time. Many efforts are being made to investigate the pathophysiological mechanism of myocardial ischemia-reperfusion injury and to search for strategy that will protect myocardium against ischemia-reperfusion injury.One of the approaches that have gained widespread recognition involves the use of ischemic preconditioning,which has displayed significant protective effects in myocardial ischemia-reperfusion injury through its abilities to stimulate endogenous protective mechanisms.However,ischemic preconditioning must be performed prior to the sustained ischemic.Its usefulness is limited in clinical practices.Recently,ischemic postconditioning has attracted extensive interest due to a novel strategy of attaining cardioprotection.Ischemic postconditioning refers to the brief episodes of ischemia at the onset of reperfusion following a prolonged ischemia.It may alleviate myocardial ischemia-reperfusion injury.Since ischemic postconditioning can be easily applied with precisely controlled timing,this approach appears to have a great potential for clinical application.Many studies about the reduction of ischemic-reperfusion injury by postconditioning has now been documented and reported in small animal models using such as rats,rabbits and canines.But the effects on pigs,a large animal species that has most similar heart to humans,are not clear yet.In addition,there are few experiences in closed-chest of larger animals.Furthermore,most of the previous studies have reported that ischemic postconditioning inhibits local informatory responses in the area at risk and reduces infarct size,as well as improves cardiac function after a short period of reperfusion(<3 h after reperfusion).We have little information about whether long-term cardioprotection effect is likely achieved by antiapoptotic mechanisms after prolonged reperfusion.Pig was used in our research.We established the closed-chest model of myocardial infarction in pigs.Based on the model,we observed ischemic postconditioning treatment on pig myocardial protection.Next,we explored antioxidant and inflammatory mediators in ischemic postconditioning against myocardial ischemia-reperfusion injury.Then,we observed and compared the effects of ischemic postconditioning on myocardial biochemical marker,infarct size,ultrastructural alterations of cardiomyocytes,myocardial apoptosis and apoptosis related-genes(Bcl-2,Bax) protein expression.This study may provide not only a novel approach for clinic application but also useful theoretical and practical evidence for reducing myocardial ischemia-reperfusion injury.Methods1.Establishment of closed-chest model of myocardial infarction in pigs.Healthy Diannan small-ear pigs were randomly divided into 2 groups.(1) sham-operated (Sham) group:only put the balloon up to the distal position of the left anterior descending artery(LDA),without ischemia;(2) ischemia-reperfusion(I/R) group:animals underwent 60min of LAD occlusion followed by 72 hrs of reperfusion without additional intervention. During the experiment,animals were monitored by heart rate,blood pressure,coronary angiography,and ECG.Myocardial biochemical marker of animals were tested,including creatine kinase(CK),lactic dehydrogenase(LDH),creatine kinase-MB(CK-MB),and troponin I(cTnI,).And the changes of myocardial histopathology were observed.2.The protective role of ischemic postconditioning against ischemia-reperfusion injury in pig myocardiumPigs were randomly divided into 4 groups.(1) Sham-operated(Sham) group;(2) Ischemia-reperfusion(I/R) group;(3) ischemic postconditioning(Postcond) group:treated the same as I/R group and 8 cycles of 30s R and 30s I before reperfusion.(4) ischemia(I) group:the 1/2 size of balloon were placed in remote LAD,resulting in the formation of autologous blood clot that caused myocardial ischemia.Detected myocardial biochemical marker included CK,LDH,CK-MB and cTnI.And myocardial infarct size and ultrastructure of myocardial cells were also detected.3.Anti-oxidation and regulation of inflammatory cytokines of Ischemic postconditioning treatment to pig myocardial ischemia-reperfusion injuryThe contents of serum malondialdehyde(MDA) and superoxide dismutase(SOD) were determined respectively by spectrophotometer.Using ELISA,concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6) and Interleukin-10(IL-10) were examined.4.The role and its mechanism of ischemic postconditioning against apoptosis in myocardial ischemia-reperfusion pigsThe apoptotic cells in the myocardium were determined with electron microscope and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescence in nick end(TUNEL) method,and apoptosis-related protein molecules(Bax and Bcl-2) were studied by immunohistochemical and Western blotting analysis.Results1.Establishment of closed-chest model of myocardial infarction in pigs.After LDA distal occluded by balloon catheter,there were no blood flow showed by coronary angiography.Typical changes in ECG happened such as the elevation of the T-wave,as a first sign,and later ST-elevation.After reperfusion of 2hrs,24hrs,48hrs,and 72hrs,CK,CK-MB,cTnI,LDH myocardial biochemical markers were significantly higher than those before ischemia.Pathological features were consistent with myocardial infarction.By coronary angiography,ECG,myocardial enzymes,methods of pathological examination,it were confirmed that closed chest model of myocardial infarction for pigs was successfully established.2.The protective role of ischemic postconditioning against ischemia-reperfusion injury in pig myocardium(1) Changes in myocardial biochemical markers:compared to I/R group,CK-MB,cTnI, CK,LDH indicators in Postcond group reduced significantly(P<0.05) after reperfusion of 24hrs,48hrs and 72hrs.But index of I group was significantly increased at corresponding time(P<0.05).(2) Changes in myocardial infarction size:there was no myocardial infarction in Sham group.The myocardial infarction size of I/R group,Postcond group and I group were 23.26±3.13%,10.89±2.02%,and 33.3±12.2%,respectively.Infarct size was significantly reduced in Postcond group than in I/R group(23.26%±3.13%Vs.10.89%±2.02%, p<0.05).(3) Ultrastructural alterations of cardiomyocytes:ischemia postconditioning treatment could effectively attenuate ultrastructural damages.The results suggested that ischemia postconditioning treatment was obviously against myocardial ischemia reperfusion injury.3.Anti-oxidation and regulation of inflammatory cytokines of Ischemic postconditioning treatment to pig myocardial ischemia-reperfusion injurySOD activity was significantly increased in Postcond group than in I/R group after reperfusion of 2hrs and 24hrs.But,MDA levels were significantly decreased(P<0.05). Meanwhile,concentration of TNF-αand IL-6 in Postcond group were significantly lower (P<0.05) after reperfusion of 2hrs and 24hrs,and IL-10 concentration became significantly higher(P<0.05).4.The role and its mechanism of ischemic postconditioning against apoptosis in myocardial ischemia-reperfusion pigs(1) Changes in pig myocardial cells:with electron microscope and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescence in nick end(TUNEL) method, we found apoptotic cells in I group,I/R group,and Postcond group,except in Sham group. Apoptotic index(AI) of infarcted zone,border zone,and non-infarcted zone were significantly lower in Postcond group than those in I/R group(P<0.05).AI of infarcted and non-infarct zone in I group were significantly higher than those in I/R group(P<0.05).(2) Changes in pig myocardial cells Bcl-2 and Bax protein expressions:there were a certain amount of Bcl-2 and Bax protein expressions in Sham group myocardial cells.Bcl -2 and Bax protein expressions in I/R myocardial infarction zone,border zone and the non-infarct area increased greatly than that in Sham group(p<0.05),as well as the Bax/Bcl-2 ratio.And compared to I/R group,there were significantly reduction in Bax protein expressions of infarction zone,border zone and the non-infarcted area in Postcond group(p<0.05).There were also significantly increase of Bcl-2 protein expression in the border zone(p<0.05),and Bax/Bcl-2 ratio decline in the border zone and non-infarcted area(p<0.05).Conclusion1.Closed-chest model of pig myocardial infarction is an ideal animal model for the research of ischemic postconditioning. 2.Ischemic postconditioning can reduce the releasing of the myocardial biochemical marker;limit the infarct size and attenuate damages of myocardial cell ultrastructure after ischemia-reperfusion injury.Ischemic postconditioning can provide a long-term protection after porcine myocardial ischemia-reperfusion injury.3.The mechanisms of ischemic postconditioning resisting pig myocardial ischemia/reperfusion injury relate to anti-oxidation which may be achieved by scavenging oxygen free radicals and inhibiting lipid peroxidation,increasing antioxidant SOD levels.4.Ischemic postconditioning reduces the production of proinflammatory cytokine TNF-α, IL-6 after myocardial ischemia reperfusion injury;increases the level of anti-inflammatory cytokine IL-10 and maybe play a myocardial protection effect by regulating the balance of inflammatory cytokines.5.The mechanisms of ischemic postconditioning against pig myocardial ischemia-reperfusion injury relate to anti-apoptosis,which may be expressed by the regulating of apoptosis-related gene Bax and Bcl-2 protein expressions.6.The research provided the theoretical and experimental basis for the clinical application of ischemic postconditioning and presented the new target for the prevention of myocardial ischemia-reperfusion injury.It has the extensive application perspective as well as important value and significance更多还原