【作者】 徐丹令；

【导师】 王克强； 孙爱军； 葛均波；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2006， 博士

【摘要】 缺血缺氧引起心肌线粒体的功能和代谢改变，在心肌缺血缺氧的发病机理中处于非常重要的地位，心肌线粒体损伤诱导的凋亡蛋白表达的过程是多因素、多环节参与的病理过程，对它致病机理的研究已成为当今的热点。蛋白质组学是“后基因组时代”蓬勃发展的技术体系新型学科群，其特点是采用高通量、高分辨率的蛋白鉴定技术，全景式研究在特定病理条件下的蛋白表达谱，由此在蛋白质水平上对疾病发生、细胞代谢等过程有更全面的认识，可获得一些传统手段无法得到的新蛋白标志物和新关键分子，极大地丰富诊断标志物的选择及对复杂致病机理的全面阐明。本研究针对大鼠心肌缺血缺氧模型的心肌线粒体，进行全蛋白质组表达谱差异分析，筛出一些有潜在应用价值的差异蛋白，并在心肌组织和心肌细胞对其中的差异分子，用特异的抑制剂和基因转染等细胞和分子生物学技术进行功能的分析和验证。第一部分大鼠心肌缺血缺氧模型的线粒体乙醛脱氢酶2(ALDH2)蛋白质分析目的：利用差异蛋白质谱识别缺血缺氧大鼠心肌线粒体代谢异常的蛋白。材料和方法：实验动物分心梗组和对照组(每组9只)。对大鼠行左冠脉前降支结扎，术后4周，以LVEF达46.4±10.9％左右作为成功建立大鼠心梗模型，将之处死后，分别取左心室：先制作电镜标本，观察形态改变；然后进行心肌线粒体抽提，利用双向凝胶电泳技术显示差异蛋白的表达谱，再进行质谱鉴定和蛋白数据库分析。发现蛋白的表达差异后，分别在心梗3d、7d、14d和28d后，用RT-PCR和western blot方法证实心肌该蛋白的mRNA和蛋白质表达随时间变化的差别。另一方面，在用酶消化法获得心肌细胞后，制作细胞的缺氧模型，测定离体心肌细胞线粒体该蛋白酶在不同缺氧时间内的活性变化。结果：大鼠心梗的心肌线粒体电镜下出现线粒体肿胀，基质变淡，嵴膜疏短破坏等病理变化。在双向凝胶电泳技术显示差异蛋白的表达谱，发现其中有3个点表达有显著差异，经胶内酶解后，行质谱鉴定和蛋白数据库分析，其中一条蛋白被证实为乙醛脱氢酶2(Aldehyde dehydrogenase 2 ALDH2)，它在大鼠缺血缺氧心肌中的表达显著下降，并随心梗时间延长，心肌ALDH2的mRNA和蛋白质的表达呈逐渐下降趋势。在细胞水平的心肌线粒体(纯度达90％以上)，用乙醛代谢法测得ALDH2的酶活性也随缺氧时间的延长而下降。结论：在缺血缺氧大鼠的心肌线粒体中乙醛脱氢酶2表达显著下调，心肌缺血缺氧与ALDH2的表达和活性下降相关。第二部分乙醛脱氢酶2(ALDH2)抑制剂daidzin对大鼠心肌细胞缺氧损伤作用的机制目的：检测乙醛脱氢酶2(ALDH2)被daidzin抑制时对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响，验证乙醛脱氢酶2(ALDH2)在此过程中所起的作用。材料和方法：在确定daidzin的最佳抑制浓度后，运用心肌细胞缺氧模型，比较大鼠心肌细胞在单独缺氧和经ALDH2特异性抑制剂daidzin预处理24h后缺氧的凋亡和MAPK信号通路的改变。酶活性检测采用乙醛代谢法、ROS的检测用C400测定，凋亡的测定通过用Hoechest 33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测，MAPK信号通路酶(ERK1／2、JNK、P38)磷酸化的改变用western blotting的方法检测。结果：60uM daidzin浓度是ALDH2酶活性被抑制而细胞无凋亡发生的最佳工作浓度。在daidzin(60uM)预处理24h后再经缺氧诱导，比单独缺氧引起的心肌细胞凋亡更为明显：表现为在Hoechest 33324染色中，细胞核溶解和核碎裂(P＜0.05)，在FACS和TUNEL的检测中，凋亡细胞明显增加(P＜0.05)，经凋亡后的死亡细胞有增加趋势但无统计学差异，同时与MAPK信号通路有关的磷酸化ERK1／2、JNK和P38的酶活性均表达增强。结论：daidzin使ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，其机理是与细胞ROS和凋亡的增加及MAPK信号通路的激活有关。ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。第三部分乙醛脱氢酶2(ALDH2)基因对大鼠心肌细胞缺氧损伤的保护作用的机制目的：通过检测转染野生和缺失型乙醛脱氢酶2(ALDH2*1／ALDH2*2)基因，对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响，进一步证明乙醛脱氢酶2(ALDH2)在此过程中所起的作用。材料和方法：运用细胞缺氧模型(同上)，比较大鼠心肌细胞在转染野生和缺失型乙醛脱氢酶2(ALDH2*1／ALDH2*2)基因再缺氧后ROS、4—HNE、凋亡、MAPK酶活性、bcl2和P53的改变，酶活性检测采用乙醛代谢法、ROS的检测用C400测定，凋亡的测定通过用Hoechest33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测，4—HNE的检测用免疫组织化学法，MAPK信号通路酶(ERK1／2、JNK)磷酸化的改变用westernblotting的方法检测，而bcl2和P53分别用RT-PCR和western blotting的方法检测。结果：转染ALDH2野生型(ALHD2*1)和缺失型(ALDH2*2)基因后再缺氧，表现为转染缺失型(ALDH2*2)的心肌细胞对缺氧性损伤的耐受性明显差于野生型(ALHD2*1)的(P＜0.05)。与运用ALDH2特异性抑制剂daidzin不同的是：转染ALHD2*1和ALDH2*2后再缺氧，都对ROS的产生和MAPK途径(ERK1／2和JNK的磷酸化)没有影响(P＞0.05)；而在转染缺失型ALDH2*2基因再缺氧组，除了有细胞凋亡的增加外(P＜0.05)：表现在Hoechest 33324染色(核溶解和核碎裂)，FACS和TUNEL检测，还有4—HNE的明显增加(P＜0.05)。从bcl2和P53的RT-PCR和western blot结果分析，转染野生型(ALHD2*1)的缺氧心肌细胞bcl2表达增高，P53表达下降，与缺失型(ALDH2*2)的结果相反。结论：4—HNE可能是细胞氧化损伤的重要作用因素。ALDH2*1可降解氧化物质4—HNE，它的抗凋亡作用不以ROS-MAPK途径为主，有可能与bcl2—P53的ROS非依赖性刺激的凋亡途径有关，ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。结论1．缺血缺氧性损伤可能与大鼠心肌线粒体中乙醛脱氢酶2(ALDH2)的表达和活性显著下降有关。2．daidzin使ALDH2酶活性降低后，心肌细胞对缺氧的耐受性下降，并对缺氧导致凋亡的易感性增加，此时细胞ROS和凋亡的增加与MAPK信号通路的激活有关。3．转染ALHD2*1和ALDH2*2对缺氧损伤的心肌细胞ROS的产生和MAPK(ERK1／2和JNK)途径没有影响。ALDH2*1可降解氧化物质4—HNE，它的抗凋亡作用不以ROS-MAPK途径为主，可能与bcl2—P53的ROS非依赖性刺激的抗凋亡途径有关。4．ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。潜在价值和创新点1．我们首次通过蛋白质组学技术发现乙醛脱氢酶2(ALDH2)在缺血缺氧心肌中表达下降，并初步证实其具有心肌细胞保护作用。2．基于ALDH2的心肌保护作用，我们推测ALDH2基因缺失型这类人群在心肌梗死或高血压后更易发心力衰竭，预后更差，同时也为临床的药物治疗提供靶点。更多还原

【Abstract】 Cardiac mitochondria plays a key role in the ischemic pathogenesis of heart failure. The apoptotic regulatory proteins mediated by injuried mitochondria have a multifactorial and multistage pathogenesis. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of heart failure have not yet been fully elucidated. Recent technological advances in proteomics allow us for the first time to examine the expression profiles at protein level on a genome-wide scale. In our study, we used 2-DE and LC- MS to identify the proteins with different expressing level in rat infarction hearts. We got a group of significant differential proteins including ALDH2 protein, which may be related to the pathogenesis of heart failure. We also drew a positive conclusion through further biological analysis and validation with the specific inhibitor and transgene, and performed a series experiments related to cell apoptosis and signal route with cellular and molecular techniques.PART ONE Aldehyde dehydrogenase 2 （ALDH2） proteomic analysis of ischemic myocardialmitochondrial protein expression in ratsAbstract Aim To determine the metabolic changes in rat ischemic heart, we applied the method of proteomics to discover the possible protein variation in rat myocardial mitochondria. Methods Myocardial infarction model was established by left anterior descending coronary artery ligation. 4 weeks after operation, we used LVEF 46.4±10.9% as myocardial infarction model. The rats were killed and their left ventricules were cut off. Those mitochondrial morphologies were observed by electron microscope, and the mitochondrial proteins were extracted. Weight-age matched rats acted as control. The mitochondrial protein expression in two groups were analyzed by two-dimensional gel electrophoresis and LC-mass spectrometry, and were identified using database comparision. After found the significant changes, RT-PCR and western blot methods would be used to prove this expression difference at the time of 3d 7d 14d and 28d’s myocardial infarction. On the other hand, we made the hypoxia model from myocytes with enzymatic digestion and their mitochondrial protein activity from mycytes was measured at different hypoxia time. Results The mitochondria showed significant pathological changes in rat ischemicheart tissue under the transmission electron microscope, including shape swelling, matrix thinning and cristea breakdown. And 3 protein spots altered significantly were found. Aldehyde dehydrogenase 2 （ALDH2） was identified with database comparision. Expression of ALDH2 decreased remarkably in ischemic heart tissue compared with control. Furthermore, we detected the ALDH2 mRNA and its protein expression of rats at 3d, 7d, 14d, 28d after LAD ligation, and the results showed it decreased gradually after operation. The decrease of ALHD2 enzyme activity also showed hypoxia-time dependant in vitro by the method of acetaldehyde metabolism （the purity of cardiac mitochondria was more than 90% under the electron microscope） . Conclusion The expression of mitochondrial ALDH2 in rat ischemic myocytes decreased and also its enzyme activity showed hypoxia-time dependant in vitro.PART TWO The mechanism study of Daidzin, inhibitor of Aldehyde dehydrogenase 2, ininjuring the rat cardiomyocytes induced by hypoxiaAbstract Aim To observe and verificate the effect of inhibited ALDH2 on cardiac myocyte induced by hypoxia, including apoptosis, MAPK signal route, and the role of ALDH2. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with or without daidzin pretreatment beyond the optional daidzin concentration. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting （FACS） and The DeadEnd^TM Fluorometric TUNEL System, and MAPK signal pathway （ERK1/2 JNK P38） by western blotting. Results ALHD2 enzyme activity of myocytes could be inhibited by daidzin （24h, 20-100 μ M） without induction of apoptosis, the most effective but no apoptotic concentrantion was 60 μ M. When exposed to hypoxia, however, the ROS and apoptisis was significantly increased in the cells pretreated with daidzin compared to those without the pretreatment （P<0.05）, also an increased MAPK phospholyrated activation, including ERK1/2, JNK and P38 （P<0.05）. Conclusion Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosisfollowing hypoxia and the increase of ROS may related to MAPK activation, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury. PART THREE The mechanism study of Aldehyde dehydrogenase 2 transgene in preventing ratcardiomyocytes from apoptosis induced by hypoxiaAbstract Aim To observe the effect of wild and mutant type of ALDH2 gene on cardiomyocyte induced by hypoxia, including apoptosis and MAPK cell signal route. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with transgene ALDH2 （wild type-ALDH2*1 and mutant type-ALDH2*2） to observe the change of ROS, 4-HNE, apoptosis, MAPK（ERK1/2 and JNK）, bcl2 and P53. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, 4-HNE by immonohistology, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting （FACS） and The DeadEnd^TMColorimetric TUNEL System, and MAPK signal pathway by western blotting, bcl2 and P53 by RT-PCR and western blot. Results myocytes had a different tolerance to hypoxia after transgene of ALDH2 wild and mutant type. The wild and mutant ALHD2 transgene （ALDH2*1/ALDH2*2） had no effect on ROS and MAPK phosphoryrated activation including ERK1/2 and JNK （P>0. 05） . But in transgenic mutant type-ALDH2*2 myocytes induced by hypoxia （compared with its inhibitor daidzin）, besides the apoptotic cells, including Hoechest 33324 staining （karyopyknosis and karyorrhexis）, FACS and TUNEL, 4-HNE increased significantly （P<0.05）. Furthermore the transgenic wild type-ALDH2*1’ induced by hypoxia, their expression of higher bcl2 and lower P53 were quite different from mutant type-ALDH2*2’s（P<0.05）. Conclusion 4-HNE might be an important factor in myocytes oxidative injury. ALDH2 Transgene had no obvious effect on ROS and MAPK cell signal route. And the anti-apoptotic function from ALDH2*1 might be related to bcl2-P53 without ROS-MAPK dependant route. Myocytes showed decreased tolerance to hypoxia after transgene of ALDH2*2 and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.Conclusion1. The expression of ALDH2 in rat ischemic hearts decreased and also its enzyme activity showed hypoxia-time dependant in vitro.2. Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis following hypoxia and the increase of ROS may related to MAPK activation.3. The transgenic ALDH2*1/ALDH2*2 myocytes induced by hypoxia had no obvious effect on ROS and MAPK（ERK1/2 and JNK） cell signal route. ALDH2*1 could degrade 4-HNE and its anti-apoptotic function might be related to bcl2- P53 dependant route without ROS-MAPK’s.4. Under the lower ALDH2 enzyme activity circumstances, myocytes showed decreased tolerance to hypoxia and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2*1 in hypoxia-induced myocardial injury.The potential application and novelty of this project1. Recent technological advances in proteomics allow us for the first time to find the decreased ALDH2 expression profiles in ischemic and hypoxia rat myocytes. And our study confirmed the ALDH2 protective function in heart.2. Those with myocardial infarction and hypertension increase the susceptivity to heart failure due to the reduction of ALDH2 activity and also have a worse prognosis. We put forward that ALDH2*1 can prevents myocytes from hypoxia injury, which might discover new possible therapeutic targets in clinic.更多还原