【作者】 程何祥；

【导师】 贾国良；

【作者基本信息】 第四军医大学 ， 内科学（心血管病）， 2001， 博士

【摘要】 研究背景与目的 细胞凋亡是一种特殊的死亡形式，是不同于细胞坏死的、由基因调控的细胞主动性“自杀”过程。长期以来，坏死被认为是心肌细胞死亡的唯一方式。近来研究表明，细胞凋亡参与了心肌缺血-再灌注损伤的病理过程，凋亡抑制基因bcl-2及调亡促进基因bax、bad、fas、p53等共同调控这一过程。细胞凋亡是缺血性心脏病领域的崭新课题。 但是关于单纯缺血/缺氧是否可以导致心肌细胞凋亡，目前尚存在争议。心肌缺血—再灌注损伤中细胞凋亡的发生机制也未完全阐明。近年来，氧自由基和钙离子在心肌缺血—再灌注损伤中的作用受到高度重视；然而尚不清楚两者在心肌缺血—再灌注损伤细胞凋亡发生中的作用。 在保护心肌缺血—再灌注损伤的各种措施中，缺血预处理（IPC）尤为引人注目。但关于IPC与心肌细胞凋亡的关系尤其K_ATP通道的作用，研究还不够。近年来牛磺酸、胺腆酮拮抗氧自由基和钙超载的作用已引起重视，但是关于两者对心肌细胞凋亡的影响，尚未见报导。阿斯匹林广泛应用于缺血性心脏病的防治，然而具有抑制心肌前列环素合成的不利作用。阿斯匹林是否会因此增加心肌细胞凋亡的发生率？明确此点对临床实践具有重要意义。 本课题将对这些问题进行系统研究，以探讨细胞凋亡在心肌缺血—再灌注损伤和氧化应激中的作用，以及采用拮抗自由基和钙超载的药物或措施抑制心肌细胞凋亡的可能性和意义。方法 以离体培养的新生SD大鼠心肌细胞，建立心肌细胞缺氧—复氧、模拟缺血—再灌注模型及过氧化氢诱导的氧化应激模型；用成年SD大鼠建立在体心肌缺血—再灌注（I-R）模型及缺血预处理（IPC）模型。分别采用牛磺酸、胺腆酮、吡那地尔、阿斯匹林等作干预因素，并随机化分组。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术（TUNEL）、流式细胞仪碘化丙啶（PI）染色法和双标法检测细胞凋亡率，用透射电镜（TEM）和荧光显微镜观察细胞形态学，琼脂凝胶电泳查DNA梯，免疫组化检测凋亡相关基因的蛋白表达（Bcl-2、Bax、Bad、P53）等作为凋 臼四军医大学博士学位论文 一 亡指标：以心肌梗死范围测定、心肌酶释放及细胞存活率计数反映心肌损伤坏死 程度；通过心肌超氧化物歧化酶pOD)活性及丙二醛（MDA）含量测定，反映氧 化水平；用激光扫描共聚焦显微镜技术测定细胞内游离ca》浓度。结果中的数据 资料用SPLM软件进行统计处理。 结果与结论 1．细胞凋亡的检测需多种方法相结合，其中 nnexin V—FITC染色法可识别 出凋亡早期的细胞（2—3h），而且能够区别细胞凋亡与坏死，有较高的特异性和 灵敏性，是目前较理想的凋亡检测方法。十 2．培养的新生大鼠心肌细胞缺氧Zh一复氧6h后，除出现坏死指标外，TU’NEL 呈现阳性染色，透射电镜发现典型的凋亡细胞，流式细胞仪PI染色法出现明确的 亚二倍体峰，Bax表达明显增强，表明缺氧一复氧诱导培养的心肌细胞发生凋亡； 大鼠在体缺血一再灌注模型也证实心肌缺血30min一再灌注6h后，Bax蛋白的表 达显著增强。因此离体及在体心肌缺血一再灌注皆可诱导心肌细胞凋亡。 3．培养的心肌细胞在单纯缺氧sh组和单纯缺氧48h＋换液组，TU’N’EL检测、 流式细胞仪、透射电镜皆未发现阳性凋亡指标，说明单纯缺氧未诱导心肌细胞凋 亡。而模拟缺血 sh组…H 68)和单纯缺氧 48h不换液组，TUN’EL检测阳性，流 式细胞仪发现亚二倍体峰，透射电镜发现典型凋亡细胞；表明急、慢性模拟缺血 可诱导心肌细胞凋亡，细胞外液酸化是心肌细胞凋亡的刺激因素。还提示进行心 肌细胞缺血／缺氧研究时，不同的模型建立方法可能会对实验结果产生较大影响： 心肌单纯缺氧模型应保持培养液的pH值稳定。 4．心肌缺血一再灌注在出现细胞凋亡的同时，伴有显著的MDA含量升高， 提示氧自由基可能是缺血一再灌注致心肌细胞凋亡的始动和关键性介质。 5．低浓度过氧化氢门)可诱导培养的心肌细胞发生凋亡，表现为 透射电镜下的特征性超微结构变化，nnexin V染色呈现较强的黄绿色荧光，琼 脂糖凝胶电泳可见“DNA！adder”，TU’N’EL检测阳性，流式细胞仪 PI染色法呈现 典型的亚二倍体峰，流式细胞议双标法检测发现较多 Annexin V”／PI”的凋亡细胞。 高浓度过氧化氢门nunow)则主要致使细胞坏死。因此，外源性羟自由基可剂 量依赖性地诱导心肌细胞凋亡或坏死。 6．Caspase－3（CPP32）的竞争性抑帘剂Z－DEVDIMK可文抗过氧化氢诱导的心 肌细胞凋亡，表明CaspaseI途径可能是OH－诱导心肌细胞凋亡的信号通路之一。 免疫组化示过氧化氢组及抑制剂组Bad及P53表达显著增加，因此过氧化氢通过更多还原

【Abstract】 Research Background and AimApoptosis is a special kind of cell death. It is an active cell "suicide" process which is regulated by the genes and distinct from cell necrosis. Necrosis has long been recognized as the unique form of cell death in myocardium. But recent researches showed that apoptosis contributes to the pathological process of myocardial ischemia-reperfusion injury,which is co-regulated by the apoptosis-inhibiting gene Bcl-2 and the apoptotsis-promoting genes bax, fas,bad and p53 etc.Apoptosis is a new issue in the field of ischemic heart disease.But at present,there are still different opinions about whether ischemia/hypoxia alone can induce myocardial apoptosis.The mechanisms of myocardial ischemia-reperfusion injury have also not been clarified.In recent years,effects of oxygen free radicals and calcium overload on myocardial ischemia-reperfusion injury have been highly regarded.But it is still not clear what roles they play in occurrence of apoptosis during myocardial ischemia-reperfusion injury.Among the various methods to protect myocardial ischemia-reperfusion injury, ischemic preconditioning(IPC) has been paid much attention to.But its correlation with myocardial apoptosis is not fully studied,especially the role of KATP channel. In recent years,antagonisms of taurine and amiodarone on oxygen free radicals and calcium overload have gained much attention.But no report was found about their effects on myocardial apoptosis.Aspirin is widely used to prevent and treat ischemic heart diseases,wheras it has an adverse effect of inhibiting the synthesis of myocardial prostacyclin. Whether aspirin can thus increase occurrence of myocardial apoptosis or not? Clarifying this issue is very important to clinical practice.This thesis will make a systematic study on these questions to discuss the roles which apoptosis plays during myocardial ischemia-reperfusion injury and oxidative stress,and to discuss the possibility and value of inhibiting myocardial apoptosis byanti-free radicals and anti-calcium overload medication or methods. MethodsNeonatal SD rat cardiomyocytes were isolated and cultured in vitro,with which to establish models of myocardial hypoxia-reoxygenation, simulated ischemia-reperfusion and oxidative stress induced by hydrogen peroxide. In vivo models of myocardial ischemia-reperiusion (I-R) and ischemic preconditioning (IPC) were established with adult SD rats. Taurine, amiodarone,pinacidil, aspirin etc were adopted as intervening factors respectively and different groups were randomized. Terminal deoxynucieotidyl transferase-mediated dUTP nick end labeling (TUNEL), propidium iodide(PI) staining and double-staining with flow cytometry were adopted to detect the apoptotic rates. Transmission electron microscope(TEM) and fluoromicroscope were used for morphology. Agarose gel electroghoresis was made for DNA ladder.Expression of apoptosis-related genes including bcl-2,bax,bad and p53 were detected by immunohistochemistry. These were used as apoptosis indicators.The extent of myocardial necrosis was reflected by detecting range of myocardial infarction, release of myocardial enzymes and the cell livability. The oxidative level was reflected by examining activity of superoxide dismutase(SOD) and quantity of malondialdehyde(MDA) in myocardium. Intracellular calcium concentration was detected by laser scanning confocal microsopy.Data in the results was statistically analized with SPLM software. Results and Conclusions1.Detection of apoptosis needs to combine several methods together,among which Annexin V-FITC stain can recognize apoptotic cells at an early phase(from the second to third hour) and can distinguish apoptosis from necrosis with high specificity and sensitivity,thus being an ideal method to detect apoptosis at present.2. After two hours of hypoxia followed by six hours of reoxygenation, as well as features of necrosis,the cultured neonatal rat cardiomyocytes showed a positive TUNEL staining,typical apoptotic cells under TEM,a clear sub-diploid peak with更多还原