【作者】 洪毅；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景与目的:紫绀型先心病是临床常见的先天性畸形,心肌慢性缺氧是此类患者共同的病理生理过程。心肌慢性缺氧适应是促进心肌细胞存活和生长的关键,但是心肌慢性缺氧适应的确切机制尚不清楚。低氧时,内质网中蛋白质正确折叠发生障碍,导致未折叠蛋白和异常折叠蛋白质的堆积,引起内质网应激(ER stress)。未折叠蛋白反应(UPR)是内质网应激时适应性代偿的重要信号通路之一。内质网应激同时能够增强细胞自噬能力。细胞自噬和UPR被认为可能是缓解内质网应激的重要途径。深入研究慢性缺氧对心肌细胞中细胞自噬和UPR的活化及可能作用,将有助于深化对心肌慢性缺氧适应的认识。本实验分别在紫绀型先天性心脏病患儿心肌组织标本中观察细胞自噬变化;在心肌组织标本和体外培养的心肌细胞慢性缺氧模型上,研究慢性缺氧对心肌细胞中UPR活性变化的影响,并探讨细胞自噬和UPR在心肌慢性缺氧适应中的意义。研究方法:本研究分三部分:第一部分:统计分析2008年在我科行先心病手术治疗患儿的临床特征和恢复、预后的关系;第二部分:选取紫绀型和非紫绀型先天性心脏病患儿,收集手术中切除的肥厚右室流出道心肌组织。透射电镜观察心肌细胞中自噬体的形成;western blotting检测LC3Ⅱ/LC3Ⅰ比值的变化,检测细胞自噬的变化;免疫组化观察GRP78/Bip在心肌组织中的表达;提取心肌组织的总RNA,采用RT-PCR检测GRP78/Bip和XBP-1s mRNA表达水平;提取心肌组织的总蛋白,应用Western blot检测XBP-1s蛋白表达水平,PERK、eEF2α磷酸化水平以及ATF6f的表达水平;第三部分:体外培养H9c2大鼠心肌细胞株,将细胞暴露于1% O2、5% CO2的环境中建立缺氧细胞模型,培养不同时间段后,提取心肌细胞的总RNA,应用RT-PCR检测GRP78/Bip和XBP-1s mRNA表达水平的变化;提取心肌细胞的总蛋白,应用Western blot检测第二部分实验指标的变化。研究结果:主要结果如下:1、紫绀型先心病患者在术前心肌酶谱、术中转流时间、术后ICU监护时间和心包引流量方面显著高于非紫绀型先心病患者。说明紫绀型先心病患者虽然在术前与非紫绀型先心病患者差异不大,但实际病情更重,预后差,恢复时间长;2、紫绀型先天性心脏病患儿心肌组织中可观察到自噬体的存在,且LC3Ⅱ/LC3Ⅰ比值显著升高,提示细胞自噬作用增强;3、与非紫绀组相比,紫绀型先天性心脏病患儿心肌组织中GRP78/Bip蛋白表达明显增强(p < 0.001)。免疫组化检测表明GRP78/Bip在胞浆中有较强的表达。相似的,GRP78/Bip mRNA表达水平显著升高(p < 0.01);4、紫绀型先心病患儿心肌组织中PERK活性和ATF6活性均显著增强(p < 0.001),但是IRE1活性无显著变化(p > 0.05);5、体外细胞实验研究发现:在缺氧条件下UPR活性表现出缺氧时间依赖性的变化。其中,IRE1在缺氧的最初6小时内升高最快,6小时左右达到高峰,此后逐渐降至正常对照水平;PERK活性在12小时左右达到高峰,此后维持在较低但高于对照水平; ATF6活性在24小时内逐渐升高,在12小时左右达到高峰,此后维持在较低但高于对照水平;结论:1.紫绀型先心病患者术前存在一定程度的心肌损害,表现为术前心肌酶谱升高。2.首次观察到紫绀型先心病患儿心肌组织中细胞自噬体形成,UPR三条信号通路激活,尤其ATF6和PERK信号通路活性增加明显。3.建立H9c2心肌细胞株慢性缺氧细胞模型;慢性缺氧时IRE-1信号通路早期活性升高,随后逐渐降低至正常水平,ATF6和PERK信号通路活性水平均出现先逐渐升高后稳定在高水平的趋势。综上所述,自噬体形成和UPR信号通路激活对慢性缺氧条件下,心肌细胞的存活和功能调节可能有重要意义。更多还原

【Abstract】 Background and Objective:Congenital heart disease, especially cyanotic cardiac defects which result in chronic hypoxia in the neonate until operative correction is achieved, remains a major cause of death in infancy. Myocardial adaptation to chronic hypoxia is critical for the survival and function of cardiomyocytes. Under hypoxic condition, the maturation of protein in the cardiomyocyte was frustrated. Many misfolded or unfolded protein accumulating in the endoplasmic reticulum (ER) triggers ER stress. Cells have developed elaborate protein quality control systems that recognize improperly folded proteins and either refold them or facilitate their degradation. One such quality control system is the unfolded protein response, or the UPR. The UPR is a highly conserved signal transduction system that is activated when cells are subjected to ER stress in ways that impair the folding of nascent proteins in this organelle. Recent observations indicate that in the heart, the UPR is activated during acute stresses, including ischemia/reperfusion. However, the roles of UPR in the longer term stresses such as chronic hypoxia have not been well defined.Autophagy is an evolutionarily conserved process that results in the degradation of cytosolic components inside lysosomes. Under normal conditions, it is a nonstop, reparative, life-sustaining mechanism for recycling cellular components, such as long-lived proteins and damaged organelles. Therefore, autophagy is thought to be involved in many physiological processes, including cellular differentiation, tissue remodeling, growth control, cell defense and adaptation to an adverse environment. Autophagy can be stimulated and this occurs as a cellular response to both extracellular (e.g. nutrient starvation, hypoxia, overcrowding, high temperature) and intracellular (e.g. accumulation of damaged or superfluous organelles) stress conditions. Also, both ER stress and the UPR could trigger autophagy. However, whether autophagy plays a protective role in cardiomyocytes under chronic hypoxia is unclear. So, the aim of this study is to provide an insight into the involvement of autophagy and the UPR in the myocardial adaptation to chronic hypoxia both in vivo and in vitro.Methods:Samples taken from the right ventricular outflow tract were collected from patients with cyanotic(n = 19) or acyanotic (n = 12) congenital heart disease. Autophagosome was studied by electromicroscopy, while the protein level of LC3Ⅰand LC3Ⅱwere evaluated by western blotting. For the activity of UPR, the expression of Bip was examined by immunohistochemistry, while XBP-1 splicing mRNA and Bip mRNA were tested by RT-PCR. The XBP-1s protein, phorspho-eIF2α, Bip, ATF6f and phorspho-PERK were tested by western blotting.To evaluated the effect of chronic hypoxia on each branch of the UPR in the cardiomyocytes in vitro, primary rat cardiomyocytes and embryonic rat-heart-derived H9c2 cells were cultivated and exposed to 1.0% O2, 5.0% CO2 for different durations to establish the chronic hypoxic cell model. Control cells were cultivated in the same conditions except for 21% O2 concentration. After different duration of hypoxic exposure, cells were collected and subjected to RT-PCR and western blot to detecting the mRNA and protein expression of the UPR.Results:1. By electron microscope, we found autophagosome in the cytoplasm.The rate of LC3Ⅱ/LC3Ⅰwas increased (p < 0.001) in the myocardium from patient with cyanotic cardiac defect.2. Immunohistochemistry revealed that GRP78/Bip was expressed in the cytoplasm. The protein levels of GRP78/Bip were significantly elevated (p < 0.001) in patients with cyanotic compared to acyanotic congenital heart disease. Also, the expression of GRP78/Bip mRNA were markedly increased in cyanotic patients (p < 0.05).3. Western blot analysis revealed that the level phospho-PERK, phorspho-eIF2αwas elevated in cyanotic hearts compared with their acyanotic counterparts (p < 0.001); also, the level of ATF6 fragment was increased (p < 0.01). However, there are no significant difference about the XBP-1 splicing mRNA and XBP-1s protein expression in this two groups (p > 0.05).4. The expression of XBP-1 splicing mRNA and XBP-1s protein in hypoxic cultivated H9c2 cells were significantly increased in the first 6h after exposed to hypoxia. And, it decreased sharply to the control level.5. In hypoxia-esposed cells, the levels of phospho-PERK, phorspho-eIF2αwere increased in the first 12h, and stayed at nearly that high level during the period of hypoxia.6. Similarily, ATF6 fragment, Bip mRNA and rotein were all increased in the 24h after exposed to hypoxia, and remained highly expression.Conclusions:Expression of autophagy was increased in patients with cyanotic cardiac defect. Upregulation of endogenous UPR in cardiomyocytes under chronic hypoxia has been observed both in vivo and in vitro. Taken together, the findings here suggested that UPR might plays an important role in myocardial adaptation to chronic hypoxia.更多还原