心血管系统与消化系统蛋白质组学研究进展

1、1,第十一讲：心血管疾病蛋白质组学与消化系统疾病蛋白质组学研究进展,重庆医科大学药学院 邱宗荫 ,2,、心血管疾病蛋白质组学研究 、脂肪组织的蛋白质组学研究 、幽门螺旋杆菌的蛋白质组学研究,3,、心血管疾病蛋白质组学,Cardiovascular Proteomics,4,心血管系统,心血管系统由心脏、血管和调节血液循环的神经体液装置所组成，其功能是通过血液将氧和营养物质供给组织，将组织代谢废物运出。 The human heart beats over 100,000 times and moves 5700 liters of blood each day throughout the l

2、ifetime of an individual.,5,心血管疾病,心脏和血管疾病（心血管疾病）是多种疾病的总称。 包括动脉粥样硬化，冠状动脉粥样硬化症，冠状动脉硬化性心脏病，血栓阻塞部分心脏内的血液流动引起的心脏病发作，高血压病，风湿病，心力衰竭，心律失常，心脏瓣膜疾病，向大脑供血的血管发生阻塞引起的缺血性中风，大脑内的血管破裂引起的出血性中风等。,6,卫生部心血管病防治研究中心：中国心血管病报告2006,中国处于经济转型期，人们的生活方式发生重大变化。总体上看，中国人口预期寿命有所延长，但以心血管病为代表的慢性病日趋增加，发病年龄前移。心血管病的高发病率、高致残率和高死亡率给社会与居民造成

3、沉重负担，已成为重大的公共卫生问题。 19902005年，中国农村居民心血管病死亡率波动160240人/10万人，城市居民为210240人/10万人，居死因构成首位。估计每年全国心血管病死亡300万人，占总死亡人数的1/3强。,7,中国心血管病危险因素水平持续升高,中国每年新增加高血压病患1000万人，估计2006年高血压人数已达2亿。 血压升高是心血管病发病和死亡的主要危险因素；血压升高与糖尿病、慢性肾脏病发生和总死亡密切相关。高血压患病率城乡差距逐渐缩小，甚至农村人群收缩压水平高于城市。 中国每年新增加血脂异常病患1000万人，估计2006年血脂异常人数已达2亿。 高胆固醇血症患病率为2.

4、9%，估计2006年全国有高胆固醇血症患者近3000万。血清总胆固醇水平升高与缺血性心血管病密切相关。,8,中国每年至少新增加超重人数1000万人，估计2006年超重人数超过2.4亿，肥胖人数达到7000万。中国儿童超重/肥胖率增长较快。超重/肥胖或腹性肥胖与高血压、糖尿病、冠心病及脑卒中发病均有关。体重指数(Body Mass Index, BMI)与总死亡及心血管病死亡存在“U”型关系。 近年来，糖尿病发病增长速度较快，估计2006年糖尿病人数超过3 000万。代谢综合征是多个危险因素的聚集体，局部地区调查患病率约10%20%。 中国15岁以上人群吸烟率为35.8%，全国烟民高达3.5亿人

5、，被动吸烟者为5.4亿。1519岁青年吸烟率呈上升趋势；开始吸烟的年龄较1984年提前了45年。,9,中国大部分农民和城市中青年居民均缺乏体力活动，体力活动不足是导致超重/肥胖、血脂异常、糖尿病、高血压的危险因素。 中国居民膳食结构不尽合理，城乡居民谷类摄入量呈减少趋势，脂肪摄入量呈上升趋势。脂肪摄入量过多是导致超重/肥胖、血脂异常、糖尿病、高血压等危险因素水平上升的重要原因。,10,Cardiovascular Proteomics,应用蛋白质组学技术从整体上分析心血管系统的蛋白质组成成分的动态变化，了解蛋白质间的相互作用，将有助于阐明心血管疾病发病的分子机制和筛选特异的生物标志物。,Pub

6、 Date:December 2006,11,Applications of proteomics in cardiovascular disease,Understanding of novel pathophysiological mechanisms. Identification of diagnostic/prognostic biomarkers for cardiovascular disease. Identification of novel therapeutic targets.,Peter P. Liu, et al. Cardiovascular Proteomics

7、: Tools to Develop Novel Biomarkers and Potential Applications. J Am Coll Cardiol, 2006; 48:1733-1741. (IF 9.701),12,临床将研究结果主要用于：,从蛋白质水平研究相关心血管疾病的发病机制； 应用心血管疾病蛋白标记物来更早、更准确地检测心血管疾病，尤其是急性冠脉综合征(ACS)； 蛋白质组学来源的信息作为目前患者诊断的补充； 蛋白质组检测获得的信息有利于个体化治疗，为其提供新的治疗靶位； 蛋白质组学检测工具和信息用于治疗的各个阶段，可以适时评估治疗的效果和校正治疗方案。,13,2-D

8、E Heart Protein Databases Accessible Via WWW,Emma McGregor, Michael J. Dunn. Proteomics of the Heart.Circulation Research. 2006;98:309,14,A 2DE separation of 80 g of heart (ventricle) proteins.,The first dimension comprised an 18-cm nonlinear (NL) pH 3 to 10 IPG subjected to isoelectric focusing. Th

9、e second dimension was a 21-cm 12% SDS-PAGE gel. Proteins were detected by silver staining. The NL pH range of the first-dimension IPG strip is indicated along the top of the gel, acidic pH to the left. The Mr scale can be used to estimate the molecular weights of the separated proteins.,15,LCM of h

10、uman cardiac tissue,A, Cardiac myocytes. B, Blood vessels. NL indicates nonlinear.,16,Proteomics-based Development of Biomarkers in Cardiovascular Disease-Mechanistic, Clinical, and Therapeutic Insights,Manuel Mayr , et al. Molecular similarly changes in human states of health are the result of chan

11、ges in the proteomes of individual patients over time in response to endogenous and/or external stimuli.(细胞, 器官, 机体的最终表型都反映在对于适时蛋白质组的描述; 处于健康状态的人体对于内源性 和/或 外源性刺激的经时性应答的变化会反映在患者个体蛋白质组的变化上面),19,The advent of novel proteomic approaches to investigate the complexity of human illness promises to shed new

12、 light on the pathogenesis of a broad range of cardiovascular diseases.(蛋白质组学途径对于在广泛范围内研究人类心血管疾病的复杂机制带来新的曙光) These inferences are multifaceted and include the commonly recognized role of proteomics in characterizing biomarkers and biosignatures for the prognosis and diagnosis of disease, the capabil

13、ity of these technologies of revealing information regarding functional subproteomes of organelles and networks in the heart and vasculature, and the importance of proteomics in defining changes in these functional subproteomes to guide future therapy.,20,PROTEOMICS AND CARDIOVASCULAR DISEASES,Prote

14、omic Analysis in Diseased Cardiac Phenotypes Vascular Proteomics Atherosclerotic(动脉粥样硬化) Cardiovascular Disease Hypertension(Animal Models; In Vitro Models) Congenital Heart Disease(先天性心脏病),21,ORGANELLE PROTEOMICS AND CARDIOVASCULAR PHENOTYPES细胞器蛋白质组学与心血管表型,The many cell types in the cardiovascular

15、system include cardiac myocytes (心肌细胞), vascular smooth muscle cells (血管平滑肌细胞), fibroblasts (纤维原细胞), and vascular endothelial cells (血管内皮细胞). Although the precise number of proteins hosted by the cardiovascular proteome is debated, the tremendous complexity of the cellular proteomes is widely recogn

16、ized and is clearly beyond the analytical capabilities of current proteomic technologies.(虽然对心血管蛋白质组中的蛋白质数量还有争议,但一致公认的是,其中的细胞蛋白质组是非常复杂的,并且以现有的蛋白质组学研究技术尚不能充分了解这种复杂性),22,Reduction of this complexity either by dividing the target into more manageable fractions or by focusing on specific organelle prote

17、omes affords an attractive alternative and, more importantly, represents an attainable goal for better spatial and functional correlations of the identified proteins.(为了降低这种复杂性,可以采取两种方式:1、把研究对象分组为较为简单的几个部分,分别进行研究; 2 、进行细胞器蛋白质组学的研究, 这种研究方式可以同时了解所鉴定蛋白质的功能与细胞内定位的关系),23,Organelle proteomic approach,In t

18、his approach, the identified proteins come with distinct subcellular locations, which are often coupled with the biological processes in which they participate , thus providing the functional significance of proteomic data. Among the essential cardiovascular cellular organelles, mitochondria(线粒体), c

19、aveolae(细胞膜穴样内陷,质膜微囊), and proteasomes(蛋白酶体) have received attention.,24,Fuctions of mitochondria：,1、oxidative phosphorylation and energy supply 2、maintenance the intracellular balance of metabolites and ions 3、protect the cell from toxitation of products of OXPHOS(氧化磷酸化) 4、play a significant role i

20、n cell necrosis and apoptosis,我也参与真核细胞的决策,并不仅仅是一台处于幕后的发动机,25,sa B. Gustafsson and Roberta A. Gottlieb. Heart mitochondria: gates of life and death. Cardiovasc Res 2007 0:cvm005v2-10,Mitochondria are important generators of energy, providing ATP through oxidative phosphorylation. However, mitochondri

21、a also monitor complex information from the environment and intracellular milieu, including the presence or absence of growth factors, oxygen, reactive oxygen species(活性氧,ROS), and DNA damage.,Mitochondria as a central integrator of multiple stress signals(胁迫信号).,26,Mitochondria have been implicated

22、 in the loss of cells in various cardiac pathologies, including ischaemia/reperfusion injury(缺血-再灌注损伤), cardiomyopathy(心肌病), and congestive heart failure(慢性充血性心力衰竭,CHF). The release of factors such as cytochrome c(细胞色素C), second mitochondria-derived activator of caspases(Smac, 第二个线粒体来源的半胱氨酸酶激活剂Smac)

23、 ，Omi/Htr2A(HtrA是线粒体丝氨酸蛋白酶家族新成员, Omi指mitochondrial intermembrane space即线粒体膜间隙), and AIF(凋亡诱导因子,apoptosis-inducing factor) from mitochondria serves to activate a highly complex and regulated cell death program. Furthermore, mitochondrial calcium overload(线粒体Ca2+超载) might trigger opening of the mitoch

24、ondrial permeability transition pore(MPTP, 线粒体通透性转运孔), causing uncoupling of oxidative phosphorylation, swelling of the mitochondria due to influx of water, and rupture of the mitochondrial outer membrane.,27,General strategy for organelle-based proteomics: example of cardiac mitochondria,BN-PAGE /S

25、DS-PAGE是一种适合于多蛋白复合物(multi-protein complexes, MPCs)分离的双向电泳技术。其第一向的BN-PAGE能使分离得到的复合物保持天然状态，因采用Coomassie Blue G250作指示剂而得名。随后的SDS-PAGE则使复合物的各组分分开，从而使各组分得到有效的分离。 BN-PAGE /SDS-PAGE电泳在线粒体呼吸链复合物、多蛋白复合物和蛋白质包涵体的研究中得到广泛应用。,28,细胞表面的穴样内陷-质膜微囊(caveolae),Caveolae是细胞表面50100 nm大小的凹陷, 含有Caveolin (一种小分子量的蛋白，21KD)。大量存在

26、于脂肪细胞、上皮细胞和平滑肌细胞。 Caveolae参与细胞的胞吞、胞饮作用、胆固醇的转运和信号转导，是细胞的信号处理中心，具有广泛的抑制信号转导的作用。另外，caveolae对血管壁脂质稳态和eNOS功能都起着重要的调节作用，其功能紊乱是动脉粥样硬化发病机制中的重要环节。 Caveolae作为细胞表面的一个重要的细胞膜结构受到了人们的普遍关注,29,Caveolae and caveolin structure.,Note the uniform size and shape of caveolae (some are indicated by arrowheads) and the num

27、erous caveolae around larger surface-connected vacuoles (asterisks).,caveolae (arrowheads) in a primary fibroblast shown in a conventional plastic section.,image at higher magnification,Sheets of plasma membrane from adipocytes labelled for caveolin-1 followed by 10 nm protein-A-gold.,30,Caveolae, a

28、 unique cellular organelle with a unique marker protein,A) Electron micrograph of an endothelial cell showing caveolae, 50-100 nm structures that are either direct invaginations or in close proximity to the plasma membrane. Caveolae are estimated to make-up an estimated 30-70% of the plasma membrane

29、 area in certain cells such as endothelial cells, adipocytes(脂肪细胞), or Type I pneumocytes(型肺泡细胞). B) Diagram comparing the biochemical composition of lipid rafts(脂筏,是膜脂双层内含有特殊脂质和蛋白质的微区。其组分和结构特点有利于蛋白质之间相互作用和构向转化，可以参与信号转导和蛋白质转运。) and caveolae (adapted from (Galbiati et al., 2001). Lipid rafts form via

30、 a coalescence of cholesterol and sphingolipids; as a result, these microdomains have vastly different biochemical properties than the bulk phospholipids bilayer. Caveolae are generally considered to be invaginated lipid rafts primarily due to an enrichment in a family or proteins known as the caveo

31、lins. Here, the caveolin oligomer is depicted as a dimer for simplicity.,31,蛋白酶体, 蛋白水解小体(proteasome),Proteasome是细胞中调节蛋白质降解最重要的细胞器，具有调控基因表达，细胞分化，细胞凋亡和信号转导等重要的生理功能，与多种疾病的发生相关。 Proteasome由1020个不同的亚基组成中空的圆桶形的结构。这些亚基在动态细胞中形成结构不一的蛋白水解小体复合物，既存在于细胞核中，又存在于胞质溶胶中。 蛋白酶体是溶酶体外的蛋白水解体系，主要降解两种类型的蛋白质：错误折叠的蛋白质和需要进行数量调

32、控的蛋白质。 蛋白酶体对蛋白质的降解通过泛素(ubiquitin)介导，所以又称为泛素降解途径。,32,Organelle proteomics in the cardiovascular system,33,Types of biomarkers/biosignatures to be used for cardiovascular disease.,There are at least three different types of biomarkers that can be developed for cardiovascular medicine, including mechan

33、istic markers, clinical disease markers, and therapeutic markers. 与心血管病医学有关的生物标志物至少可以分为三种类型: 机制标志物, 临床疾病标志物, 治疗标志物。,34,Mechanistic markers,In the first group, changes in the subcellular phenotype of the organism can lead to alterations in proteins detectable as markers. These changes, constituting m

34、echanistic markers, are closely reflective of what is going on in the cell and how the signaling pathways are manipulated. 机制标志物是指机体亚细胞表型发生改变时出现的可检测的蛋白质变化。它们反映出细胞的病理变化以及与之相关的信号通路的变化。,35,Clinical disease markers,Second, the arrival of disease carries with it changes in proteins that are detected by p

35、roteomics, so-called clinical disease markers. These markers are specific to the individual disease state and can indicate the state of progression, severity, and location of the syndrome. 临床疾病标志物是指可通过蛋白质组学方法检测的，伴随疾病而来的蛋白质变化。它们具有与疾病个体相关的特异性，能指示出疾病的进程、严重程度和症状部位。,36,Therapeutic markers,Lastly therapeu

36、tic markers are those that become evident as the treatment of a chronic disease progresses in the patient. These markers are influenced by many factors, including individual nature of the disease, drug treatment, patient activities, etc. 治疗标志物反映慢性病治疗进程。这些标志物受多种因素影响,如疾病个体的性质、治疗药物、患者行为等。,37,这些标志物的变化可用

37、 于调整临床处理方案,这些标志物可以指示出与 治疗有关的亚细胞机制,这些标志物有助认识疾病的分子起源, 提示调节表型的细胞治疗方向,38,案例:慢性心肌缺血中的自噬作用,Lin Yan, et al. Autophagy in chronically ischemic myocardium Proceedings of the National Academy of Sciences (PNAS), September 27, 2005 vol. 102 no. 39 1380713812,39,细胞的死亡,人体内的细胞注定是要死亡的，有些死亡是生理性的，有些死亡则是病理性的，有关细胞死亡过程

38、的研究，近年来已成为生物学、医学研究的一个热点 多数学者将细胞的死亡形式分为凋亡性程序性细胞死亡(Apoptosis)、自噬性程序性细胞死亡(Autophagy)和细胞坏死(Necrosis)三种类型。,Mary C, Abraham and Shai Shaham. (2004) Death without caspases, caspases without death. TRENDs Cell Biol 14(4):184-193.,40,自噬与凋亡,自噬-“自己吃自己”，是凋亡之外的第二种程序性细胞死亡方式。细胞的自噬被认为是参与绝大多数长半衰期蛋白质的降解。 自噬一旦被过分激活，就会

39、导致自噬性细胞死亡（Autophagic Cell Death），这是有别于凋亡的另一种程序性细胞死亡。,凋亡也称为型程序性细胞死亡，是细胞的一种基本生物学现象，在多细胞生物去除不需要的或异常的细胞中起着必要的作用。 细胞凋亡是机体清除多余、变异或恶化细胞的一种主动、程序化生理过程。该过程的正常与紊乱与机体进行新陈代谢、保持自身相对稳定以及肿瘤发生等密切相关。,41,Progression of cell apoptosis, visualised with Annexin V-Oregon Green.,Apoptosis of WBC,凋亡细胞的典型形态学特点表现为：细胞皱缩、体积缩小；部

40、分细胞器、核糖体和核碎片被细胞膜包裹形成凋亡小体，从细胞表面出芽脱落，最后被具有吞噬功能的细胞如巨噬细胞、上皮细胞等吞噬；磷脂酰丝氨酸外翻；细胞核染色质浓缩、边缘化、染色质DNA断裂。,42,自噬与自噬性细胞死亡,自噬(Autophagy)是指从粗面内质网的无核糖体附着区脱落的双层膜包裹部分胞质和细胞内需降解的细胞器、蛋白质等成分形成自噬体(autophagosome)，并与溶酶体融合形成自噬溶酶体，降解其所包裹的内容物，以实现细胞本身的代谢需要和某些细胞器的更新。 自噬是进化过程中高度保守的自我保护机制，是营养缺乏时细胞的适应性反应，是细胞内的再循环系统，可为生物合成提供循环利用的原料。 自

41、噬作为一种细胞生存的机制，在很多生理过程中发挥着重要的作用。自噬受许多信号通路，包括TOR、PI3K、Beclin 1及Ca2+等的调控。,43,哺乳动物细胞autophagy的三种主要方式 ：,根据细胞内底物运送到溶酶体腔方式的不同，哺乳动物细胞autophagy 可分为三种主要方式 ： 大自噬（Macroautophagy）：由内质网来源的膜包绕待降解物形成自噬体，然后与溶酶体融合并降解其内容物。 小自噬（Microautophagy）：溶酶体的膜直接包裹长寿命蛋白等，并在溶酶体内降解。 分子伴侣介导的自噬 (Chaperone-mediated autophagy，CMA)：胞质内蛋白结

42、合到分子伴侣后被转运到溶酶体腔中，然后被溶酶体酶消化。,44,错调的自噬作用与多种疾病有关,自噬可能对许多疾病包括心肌缺血/再灌注损伤、神经退行性疾病、肿瘤、感染等的发病机制和防治策略提出新的观点。 慢性心肌缺血过程中自噬性保护作用可能是“聪明心肌”的一种应对策略，合理调控心肌细胞的自噬作用，有助于维持心肌细胞稳态、减少细胞缺失。 在2008年10月8日出版的细胞代谢（Cell Metabolism）上，两个国际研究小组证实胰腺细胞自噬与糖尿病有关。,45,Hye Seung Jung, Myung-Shik Lee. Cell Metabolism, 2008, October Vol 8,

43、 318-324 Chie Ebato, Hirotaka Watada. Cell Metabolism, 2008, October Vol 8, 325-332,（封面图片：背景图片显示了高脂肪饮食小鼠的胰腺组织的电子显微镜图像，其中表现出一个自噬体螯合线粒体和胰岛素颗粒的过程。下面右侧的免疫组织化学图像显示了缺少自噬相关基因atg7的胰岛细胞存在损伤标志物p62（绿色部分）的聚集。其中红色部分为胰岛素，蓝色部分为细胞核。左侧图像显示出atg7-/-胰岛当中p62内含体的发展过程。图像提供：Ebato等）,46,胰腺细胞自噬与糖尿病的关联,来自韩国和日本的一组科学家利用了一种特异性去除A

44、tg7（自噬相关7）基因胰腺细胞的小鼠，Atg7变异的小鼠表现出葡萄糖耐量减低（impaired glucose tolerance）以及血清胰岛素浓度的降低。由于细胞凋亡的增加以及细胞分裂增生程度的降低，导致细胞数量和胰腺中胰岛素的减少。生理学分析显示，在小鼠中存在血糖激发的胰岛素分泌的降低，并且自噬作用缺陷的细胞中血糖诱导的胞内钙离子瞬变过程也被破坏。而形态学分析则表明存在泛素蛋白（ubiquitinated protein）的聚集，这种现象与p62共存，并伴随着线粒体肿胀、内质网膨胀以及细胞液泡的变化。 以上研究结果表明, 自噬作用对于维持胰腺细胞的结构、数量以及功能都是非常重要的，自噬

45、作用的破坏将导致胰岛素的缺乏以及高血糖症的发生。,47,另一篇文章中，来自日本的一组科学家称他们发现了糖尿病db/db小鼠以及高脂饮食C57BL/6小鼠的胰腺细胞中存在自噬作用的升调节。而能导致糖尿病相关的外周胰岛素抵抗的游离脂肪酸（free fatty acid FFA）诱导胰腺细胞自噬作用发生。对于atg7基因的消除将导致胰岛退化、胰岛素分泌以及葡萄糖耐量降低。与此同时研究人员还观察到，尽管在对照组小鼠中高脂肪含量的饮食能激发胰腺细胞自噬，但是对于自噬不足变异小鼠而言，这将形成葡萄糖耐量的恶化，以上现象发生的部分原因在于缺乏胰腺细胞数量的补偿性增加。 因此，基础自噬作用对于维持正常的胰岛结

46、构和功能至关重要。当存在高脂肪饮食诱导的胰岛素抵抗时，自噬作用是一种胰腺细胞的独特适应性反应。,48,Autophagy, a membrane trafficking process leading to lysosomal degradation, is implicated in cancer, neurodegeneration, and aging.,细胞的自噬过程,在形态学上，即将发生自噬的细胞胞浆中出现大量游离的膜性结构，称为前自噬泡。前自噬泡逐渐发展成为由双层膜结构形成的自噬泡，其中包裹着变性坏死的细胞器和部分细胞浆。 自噬泡的外膜与溶酶体膜融合，内膜及其包裹的物质进入溶酶体腔

47、，被溶酶体中的酶水解。此过程使进入溶酶体中的物质分解为其组成成分，并被细胞再利用，这种吞噬了细胞内成分的溶酶体被称为自噬溶酶体。,自噬泡,Autolysosomes 自噬溶酶体,49,自噬过程的形态学观察,(B) Starvation-induced autophagosomes(AP,自噬体) and autolysosomes (AL,自噬溶酶体 ) in the fat body (the functional analogue of the liver) of a fruit fly larva. Note that APs contain intact cytoplasm, wher

48、eas the contents of ALs show various stages of degradation. (C) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene, labeling cup-shaped and ring-shaped structures that correspond to IMs and autophagosomes, respectively.,Autophagy/xenophagy(异体吞噬),Autophagy “eat onself” Xenophag

49、y “eat foreign matter” Highly conserved and regulated process that maintains cellular homeostasis and protects cells against starvation and microbe invasion,The parasite Toxoplasma gondii, stripped of its protective membrane, is enveloped by a double-membraned sac and ready to be consumed by lysosom

50、es. The “surprisingly aggressive” process is called autophagy. Credit: David Ferguson, Oxford University,51,Xenophagy,可以说，自然界是节约型的（nature is thrifty），我们正在自噬作用中验证这一理论。最近两年的实验证明自噬作用可以杀死病毒和细菌，现在我们可以说自噬作用还可以杀死寄生虫。”自噬作用可以消灭致病性入侵者的新发现的功能。-George Yap, et al. Death of a Parasite: Stripped, Surrounded, Consu

51、med. Journal of Experimental Medicine.,52,Autophagy in chronically ischemic myocardium,1、动物模型的制作 2、自噬形态学的电镜观察 3、蛋白的提取 4、双相电泳分离 5、MALDI TOF MS分析 6、数据库搜索 7、差异表达蛋白的WB验证与定位研究,53,动物模型的制作,Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes

52、 of 90 min of coronary stenosis(冠状动脉狭窄) 30% reduction in baseline coronary flow followed by reperfusion(再灌注) every 12 h with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by 37%.,54,Electron micrographs of different types of AVs observed in t

53、he six-episode chronically IS region (AC) and six-episode NI region (D).,(A) Autophagic vacuoles(Avs,自体吞噬泡) containing remnants of mitochondria are demonstrated. (B) Doublemembrane AVs containing recognizable cytoplasmic contents are displayed. (C) AVs containing multivesicular bodies surrounded by

54、a sequestering membrane are demonstrated. (D) These AVs were not observed in the six-episode NI. Arrows indicate AVs. (Magnifications: 1,4002,000, AD; 5,000, A and B Insets.),55,Alteration of cathepsin D in chronically IS myocardium.,(Upper) 2D gel map of protein extracts from chronically IS region

55、after six episodes. (Lower)A magnified gel region from the NI region (Left) and the IS after either three (Center) or six (Right) episodes of CS. Arrows indicate the spots after six episodes that were all later identified as cathepsin D. MW, molecular weight. Each lower image is a 6-fold magnificati

56、on from the original gel.,56,Comparison of expression level of cathepsin B and D in chronically IS myocardium at different episodes,Comparison of expression level of cathepsin B and D in chronically IS myocardium at different episodes. (A and B) Western blot analysis of cathepsin D (A) and cathepsin

57、 B (B) at six episodes chronically IS vs. NI regions (Upper) and three episodes (Lower). The expression of cathepsins B and D significantly increased at six episodes (*, P 0.01; *, P 0.05 vs. NI). (C) The direct comparison of levels of cathepsin B (CB) and cathepsin D (CD) in chronically IS in one,

58、three, and six episodes, again showing marked increased expression of both proteins after six episodes. (D) The elevated expression of cathepsins B and D was confirmed to occur in myocytes from the chronically IS myocardium. ADU, arbitrary densitometric units.,57,Enzyme activity assays of cathepsin

59、B (A), cathepsin D (B), and -hexosaminidase (C),Enzyme activity assays of cathepsin B (A), cathepsin D (B), and -hexosaminidase (C). Activity of both cathepsin B and D and -hexosaminidase was much higher at six episodes in chronically IS region vs. NI region compared with three episodes vs. NI. *, P

60、 0.01 vs. NI; *, P 0.05 vs NI.,58,Western blot analysis of Hsc73 at six episodes chronically IS vs. NI(non-IS) myocardium (A) and at three episodes in isolated myocytes (B) and comparing effects in necrotic tissue after myocardial infarction (IF) vs. NI regions (C).,The expression of Hsc73 increased