【作者】 向飞；

【导师】 黄跃生；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 博士

【摘要】 严重烧伤后,在毛细血管通透性增加导致有效循环血容量显著减少之前,即可出现心肌缺血缺氧损害及心脏泵血功能减弱。这种伤后早期出现的心肌损害,不仅易引起心功能不全,还可诱发或加重休克,成为烧伤后早期机体缺血缺氧的重要启动因素之一。研究表明缺氧不仅在烧伤早期心肌损害的发生过程中有着重要的作用,而且也参与了多种心脏疾病的病理生理过程。因此,研究缺氧条件下心肌细胞的损害机制对于阐明严重烧伤等疾病引起的心肌损害的发生机制有着重要的意义。线粒体是细胞能量生成的主要场所,其也参与调控细胞的凋亡和坏死。在缺氧条件下,心肌细胞线粒体最易受到损伤,其结构破坏和功能紊乱直接导致了缺氧心肌细胞的死亡。研究发现缺氧等有害刺激可以引起线粒体基质肿胀,膜电位下降,外膜破裂,导致其通透性转换孔(mitochondrial permeability transition pore, MPTP)开放增加,细胞色素c (cytochrome c,Cytc)等促凋亡物质释放进入胞液,激活线粒体依赖的凋亡途径,最终造成细胞的死亡。MPTP大量开放导致的线粒体不可逆损伤是心肌细胞凋亡和坏死发生过程中的关键环节。MPTP是贯穿于线粒体内外膜由多种蛋白质组成的一个复合蛋白通道,其确切成分目前还存在着争议,但越来越多的研究认为亲环素D(cyclophilin D, CypD)是其中的一个重要调节蛋白。CypD被证实介导了多种因素引起的MPTP开放过程,抑制CypD的肽酰脯氨酰顺反异构酶的活性能够阻断MPTP的开放。肿瘤坏死因子受体相关蛋白1(tumor necrosis factor receptor-associated protein 1,TRAP1)是一个分布在线粒体,属于热休克蛋白90 (heat shock protein 90,HSP90)家族,分子量约为75KDa的分子伴侣。与典型的HSP90家族相比,TRAP1有着相似的ATP酶活性,其酶活性能够被HSP90特异性抑制剂格尔德霉素抑制,但其在调节线粒体功能方面又有着独特的作用。既往研究表明,缺氧能够诱导细胞HSP90表达增加,并且这种变化对细胞缺氧性损害起着保护作用,但缺氧后心肌细胞TRAP1的变化趋势及作用目前还不清楚。TRAP1分布广泛,在维持线粒体的自我稳定状态、调控肿瘤细胞凋亡和坏死的过程中起着重要的作用。由于MPTP的开放是细胞凋亡和坏死发生过程中的关键环节,并且进一步研究证实TRAP1在肿瘤细胞中与MPTP的重要调节蛋白CypD存在着相互作用。因此我们推测:缺氧后心肌细胞TRAP1的变化能够通过作用于CypD来影响MPTP的开放,从而发挥细胞保护作用。本研究旨在对此假设进行验证。一、研究目的:探讨缺氧后心肌细胞TRAP1的表达对细胞缺氧性损害的保护作用及其调控机制。二、材料方法:1.原代培养SD大鼠乳鼠心肌细胞,建立细胞缺氧模型。2.应用免疫印迹及免疫荧光技术观察常氧及不同缺氧时间处理后(缺氧1h,3h,6h和12h)心肌细胞TRAP1的表达变化。然后通过构建腺病毒载体转染心肌细胞干预TRAP1的表达,观察其对缺氧心肌细胞(缺氧6h)的细胞活力和死亡的影响,从而明确缺氧后心肌细胞TRAP1表达的变化趋势以及这种变化对细胞缺氧损害的作用。3.选用能反映MPTP开放的线粒体膜电位和细胞溶质中Cytc的含量作为检测指标,分别观察干预TRAP1后缺氧心肌细胞这两个指标以及caspase 3活性的改变,明确TRAP1能够调节缺氧心肌细胞MPTP的开放,并进而影响线粒体依赖的凋亡途径。在此基础上运用MPTP特异性阻断剂CsA阻断MPTP开放,探讨其能否阻止下调TRAP1表达对缺氧心肌caspase 3活性、细胞活力和死亡的作用,以此来阐明TRAP1通过减少MPTP开放,调节线粒体依赖的凋亡途径,从而发挥对缺氧心肌细胞的保护作用。4.运用免疫共沉淀和蛋白质谱分析技术明确TRAP1与CypD是否存在相互作用,同时检测干预TRAP1的表达对心肌细胞CypD蛋白和基因表达的影响。此外观察单纯缺氧以及MPTP开放抑制剂CsA对CypD蛋白和基因表达的影响,从而明确TRAP1可能是通过作用于CypD来调节MPTP开放。三、主要结果1.心肌细胞缺氧处理1h后, TRAP1的表达较常氧状态下即增加,并且随着缺氧时间的延长,TRAP1增加越明显,至缺氧后12h,TRAP1的表达与常氧组相比仍显著增多。2.缺氧6h后,过表达TRAP1减轻了缺氧导致的心肌细胞活力降低和死亡增加,而下调TRAP1表达则加重了缺氧引起的上述细胞损伤。3.缺氧6h后,过表达TRAP1减轻了缺氧导致的线粒体膜电位下降、细胞溶质中Cytc含量及胞浆中caspase 3活性增加,而下调TRAP1表达则加重了缺氧引起的上述损伤。使用MPTP开放特异性阻断剂CsA能够阻断下调TRAP1表达引起的缺氧心肌细胞活力下降、细胞死亡和caspase 3活性增加。4.常氧及缺氧条件下,心肌细胞中TRAP1和CypD均不存在着相互作用。过表达TRAP1能够使常氧心肌细胞CypD mRNA的表达增加,而下调TRAP1表达则降低了CypD mRNA的表达水平,但干预TRAP1表达对CypD蛋白含量没有明显作用。缺氧6h后,心肌细胞CypD mRNA的表达量较常氧组显著降低,而CsA在常氧和缺氧条件下均能增加CypD mRNA的表达量。同样缺氧及CsA处理后,心肌细胞CypD蛋白表达水平没有明显改变。四、结论1.缺氧能够诱导心肌细胞TRAP1表达增多,并且随着缺氧时间的延长,TRAP1的增多越明显。2.缺氧后心肌细胞TRAP1的增多对细胞缺氧性损害起着保护作用,是心肌细胞的一种内源性保护反应。3.TRAP1通过减少缺氧心肌细胞MPTP的开放,阻断缺氧诱导的线粒体依赖凋亡途径的激活,从而发挥细胞保护作用。4.TRAP1调节缺氧心肌细胞MPTP开放的机制可能是通过作用于MPTP的调节蛋白CypD来实现的。更多还原

【Abstract】 Myocardial ischemic/hypoxic damage and cardiac functional impairment precede the significant decrease in blood volume that occurs after a severe burn. In the early stages following a burn （before the decrease in blood volume）, myocardial damage not only causes cardiac insufficiency but also induces or aggravates burn shock, which can cause or aggravate ischemic/hypoxic injury to other organs. Studies have demonstrated that hypoxia is one of the main causes of myocardial damage after a burn. Hence, it is important to protect cardiomyocytes from hypoxic damage. Mitochondria are the primary target of hypoxic damage in cardiomyocytes. Mitochondrial dysfunction in hypoxic cardiomyocytes can directly lead to cell death. Following hypoxia, the opening of mitochondrial permeability transition pore （MPTP） leads to the release of apoptogenic proteins （i.e., cytochrome c） into the cytoplasm, and the activation of caspase-dependent apoptotic pathways; these steps result in cell necrosis and apoptosis. The irreversible mitochondrial injury caused by MPTP opening is critical to induce the hypoxic damage of cardiomyocytes. MPTP is a nonspecific pore consisting of several proteins. Some models have proposed the presence of other molecular components of the pore, but there is still no consensus as to the exact components. However, cyclophilin D （CypD） is generally accepted as a critical regulatory component of MPTP and plays an important role in regulating MPTP opening.Tumor necrosis factor receptor-associated protein 1 （TRAP1） localises to the mitochondria and its targeting sequence has been found in the N-terminus of the protein. An analysis of the cDNA sequences reveals that TRAP1 is identical to heart shock protein 75 （HSP75）, which is a member of the HSP90 family. The ATPase activity of TRAP1 is inhibited by Geldanamycin, which is a specific inhibitor of HSP90. Despite its ATP-binding activity, TRAP1 seems have specific functions that are different from those of other well-characterised HSP90 homologues. After hypoxia treatment, HSP90 expression increases, and it plays a protective role against damage. However, the changes induced in TRAP1 expression in the cardiomyocytes under hypoxic conditions are still unclear. TRAP1 is a mitochondrial chaperone that is critical for maintaining mitochondrial homeostasis and regulating apoptosis and necrosis of tumor cells. Furthermore,there is an interaction between TRAP1 and CypD in mitochondria of tumor cells. Since MPTP is recognized as a key player in cell death, so we hypothesise that TRAP1 protects cardiomyocytes from hypoxic damage by regulating MPTP opening, and CypD may be the target protein through which TRAP1 modulates MPTP opening. The present study is designed to test this hypothesis.Objectives:The purpose of this study was to observe the changes in TRAP1 expression after hypoxia treatment and investigate the protective effect and mechanism of TRAP1 on hypoxic damage in primary cultured cardiomyocytes.Methods:1. Primary cultured cardiomyocytes were prepared from neonatal Sprague-Dawley rats （days 1 to 3） and hypoxic conditions were established by using an anaerobic jar and a vacuum glove box filled with a mixed gas containing 94% N₂, 5% CO₂, and 1% O₂.2. TRAP1 expression was detected by western blot and immunofluorescence in cardiomyocytes under normoxic conditions and hypoxic conditions （for times of 1, 3, 6, and 12 hours respectively）. Then, a recombinant adenovirus vector for TRAP1 overexpression and a recombinant adenovirus vector for silencing of TRAP1 expression were constructed. To evaluate the role of TRAP1 changes in cardiomyocytes under hypoxic conditions, we investigated the effects of TRAP1 overexpression or TRAP1 depletion on cell viability and cell death after 6 hours of hypoxia in cardiomyocytes.3. The role of TRAP1 on MPTP opening in hypoxic cardiomyocytes was observed by investigating the effects of TRAP1 overexpression or TRAP1 depletion on mitochondrial membrane potential （Δψ） and cytochrome c content in cytosol fraction. Meanwhile, the changes of caspase 3 activity in cardiomyocytes were examined. We next investigated the effects of TPAP1 depletion on cell viability, caspase 3 activity and cell death by inhibiting MPTP opening using cyclosporine A （CsA） in hypoxic cardiomyocytes.4. Mass spectrography analysis and co-immunoprecipitation were used to determine whether there was an interaction between TRAP1 and CypD in cardiomyocytes. Then, we investigated the effects of TRAP1 overexpression or TRAP1 depletion on CypD expression both in protein levels and mRNA levels under normoxic conditions. Meanwhile, we also observed the roles of hypoxia and CsA on CypD protein expression and mRNA expression.Results:1. Western blot and immunofluorescence showed that TRAP1 content increased after 1 hour of hypoxia and continued to increase until for up to 12 hours compared with the normoxic group. Meanwhile, longer hypoxic treatments yielded higher TRPA1 expression2. After 6 hours of hypoxia, there were an increase in cell death and a decrease in cell viability. Overexpressing TRAP1 prevented hypoxia-induced damage to cardiomyocytes, while silencing TRAP1 expression aggravated an increase in cell death and a decrease in cell viability in hypoxic cardiomyocyte.3. Hypoxia caused a decrease inΔψand increased cytochrome c content in cytosol fraction and caspase 3 activation. Overexpressing TRAP1 abolished hypoxia-induced effects to cardiomyocytes and silencing TRAP1 expression aggravated hypoxia-induced damage. Furthermore, the cell damage induced by the silencing of TRAP1 was prevented by MPTP inhibitor CsA in hypoxic cardiomyocytes.4.There wasn’t an interaction between TRAP1 and CypD both in normoxic conditions and hypoxic conditions. Overexpression TRAP1 increased mRNA levels of CypD and silencing TRAP1 decreased its mRNA levels in normoxic cardiomyocytes. However, there was no effect of TRAP1 on CypD protein levels. Furthermore, hypoxia decreased CypD mRNA levels and CsA increased its mRNA levels, while they also had no effect on CypD protein levels.Conclusions1. Hypoxia treatment （for times of 1, 3, 6, and 12 hours respectively） induced a time-dependent increase in the levels of TRAP1 protein. 2. Hypoxia-induced TRAP1 increase was a protective role against hypoxic damage to cardiomyocytes.3. TRAP1 regulated the activation of mitochondrial-dependent apoptotic pathway by modulating MPTP opening in hypoxic cardiomyocytes, and then played a protective role.4. The mechanism that TRAP1 regulated MPTP opening might be though influencing CypD expression.更多还原