NEDD4蛋白调控AMPK活性参与心肌慢性缺氧适应的研究

发布时间：2018-05-23 10:28

本文选题：腺苷酸激活蛋白激酶 + 神经前体细胞表达的发育下调基因4蛋白　；参考：《第三军医大学》2015年硕士论文

【摘要】：目的:慢性缺氧是多种心脏疾病共同的病理生理过程,临床常见的疾病包括紫绀型先天性心脏病、冠状动脉粥样硬化性心脏病、代偿性心肌肥厚、高原性心脏病等。在慢性缺氧过程中,心肌细胞面临严重能量应激、氧化应激、钙离子失衡等多种应激。应激作用导致心肌细胞自身结构重塑、体液内环境改变等多种适应性改变。既往研究证实,这些适应性反应可促使心肌细胞耐受多种外界刺激。目前,心肌细胞慢性缺氧适应的确切机制尚不完全清楚。能量代谢调节是心肌细胞慢性缺氧适应的关键环节。腺苷酸激活蛋白激酶(AMP-activated Protein Kinase,AMPK)是一种在真核细胞中广泛存在的丝氨酸/苏氨酸蛋白激酶,活化AMPK可调节多种信号通路增加心肌细胞能量的生成。AMPK被认为是心肌细胞能量代谢调节的关键激酶。神经前体细胞表达的发育下调基因4蛋白(Neural precursor cell expressed developmentally down-regulated protein 4,NEDD4)是一种含有HECT结构域的E3泛素连接酶,其结构中的WW结构域可能通过识别活化AMPK结构中的磷酸化苏氨酸残基而参与其活性调节。深入研究慢性缺氧时心肌细胞中NEDD4蛋白表达水平和AMPK活化水平的变化以及NEDD4蛋白对AMPK活性的可能调节作用,将有助于我们拓展对AMPK活性调节的认识。本研究以先天性心脏病患儿心肌组织和培养H9c2心肌细胞作为研究对象,观察在慢性缺氧条件下心肌细胞中NEDD4蛋白表达水平和AMPK活化水平的变化。同时,利用小干扰RNA(small interfering RNA,siRNA)技术构建NEDD4蛋白低表达细胞模型,观察干扰NEDD4蛋白表达对心肌细胞缺氧耐受的影响。旨在通过上述实验探讨NEDD4蛋白对AMPK活性的调节作用对心肌慢性缺氧适应的影响。方法:1.选取于2013年1月-2014年1月之间在第三军医大学新桥医院心血管外科接受手术治疗的先天性心脏病患儿,根据患者术前血氧饱和度分为紫绀组和非紫绀组,取术中切除的肥厚右室流出道心肌组织作为临床标本,免疫组化染色观察NEDD4蛋白在心肌组织中的表达分布情况;ripa裂解法提取心肌标本中的总蛋白,采用westernblot法检测两组患儿心肌组织中nedd4蛋白表达水平及ampk活化水平变化。2.在慢性缺氧条件下培养h9c2心肌细胞。在不同缺氧时间段提取心肌细胞总蛋白,采用westernblot法检测nedd4蛋白表达水平和ampk活化水平变化。同时将以h9c2心肌细胞nedd4蛋白基因为靶点的sirna转染至心肌细胞,建立nedd4蛋白低表达细胞模型,在慢性缺氧条件下(94%n2,5%co2,1%o2)培养72h后westernblot法检测nedd4蛋白表达水平与ampk活化水平变化,流式细胞术检测心肌细胞凋亡情况。结果:1.紫绀组先天性心脏病患儿术前氧饱和度明显低于非紫绀组(p0.05),免疫组化检测提示nedd4蛋白主要在心肌细胞胞质中表达。与非紫绀组相比,紫绀组先天性心脏病患儿心肌组织中nedd4蛋白表达水平较低(p0.05),nedd4蛋白表达水平与患者术前血氧饱和度呈正相关。2.westernblot结果显示,与非紫绀组患儿相比,紫绀组先天性心脏病患儿心肌组织中ampk活化水平(p-ampk/ampk蛋白条带灰度比值)显著增强(p0.05)。3.在慢性缺氧条件下培养h9c2心肌细胞,westernblot结果证实,nedd4蛋白表达水平随着缺氧时间的延长而降低(p0.05),至缺氧24h时降至最低值,此后维持这一低水平表达;ampk活化水平随着缺氧时间的延长而显著增加(p0.05),在缺氧48h时达最大值,此后维持这一高活化水平;nedd4蛋白表达水平与ampk活化水平变化趋势呈负相关。4.流式细胞术结果提示:与常氧组相比,缺氧组心肌细胞凋亡比例显著增加(p0.05),但干扰nedd4蛋白表达后,缺氧所致心肌细胞凋亡比例降低(p0.05);westernblot结果提示,与阴性转染组相比,干扰转染组nedd4蛋白表达水平显著降低(p0.05),而ampk活化水平显著增强(p0.05)。结论:1.与非紫绀型先天性心脏病患儿相比,紫绀型先天性心脏病患儿心肌组织中nedd4蛋白表达水平较低,ampk活化水平增强。2.在慢性缺氧条件下培养h9c2心肌细胞,与常氧组相比,慢性缺氧组心肌细胞中nedd4蛋白表达水平降低,ampk活化水平增强。3.慢性缺氧增加心肌细胞凋亡,利用sirna下调心肌细胞nedd4蛋白表达后,ampk活化水平增强,缺氧所致的心肌细胞凋亡比例降低,这提示在慢性缺氧条件下NEDD4蛋白可能通过调控AMPK活性参与心肌慢性缺氧适应。
[Abstract]:Objective: chronic hypoxia is a common pathophysiological process of various heart diseases. The common clinical diseases include cyanotic congenital heart disease, coronary atherosclerotic heart disease, compensatory cardiac hypertrophy, and high altitude heart disease. In the process of chronic hypoxia, myocardial cells face severe energy stress, oxidative stress, calcium ion imbalance, and so on. A variety of stress, stress causes the remodeling of cardiac myocytes and changes in the environment of the body fluid. Previous studies have confirmed that these adaptive responses can induce cardiac myocytes to tolerate a variety of external stimuli. The exact mechanism of chronic hypoxia adaptation is not completely clear. The key link of chronic hypoxia adaptation. AMP-activated Protein Kinase (AMPK) is a serine / threonine protein kinase widely existed in eukaryotic cells. Activation of AMPK can regulate a variety of signaling pathways to increase the generation of myocardial cell energy,.AMPK is considered to be the key excitation of the energy metabolism regulation of cardiac myocytes. The developmental down-regulation gene 4 protein (Neural precursor cell expressed developmentally down-regulated protein 4, NEDD4) is a ubiquitin ligase containing HECT structure domain, and the WW domain in its structure may be involved in the activity regulation by identifying the phosphorylated threonine residues in the activated AMPK structure. The changes in the expression level of NEDD4 protein and the level of AMPK activation in cardiac myocytes and the possible regulatory role of NEDD4 protein on AMPK activity during chronic hypoxia will help us to expand our understanding of the regulation of AMPK activity. In this study, myocardial tissue and cultured H9c2 cardiomyocytes in children with congenital heart disease were studied. The changes in the expression level of NEDD4 protein and the activation level of AMPK in the cardiomyocytes of chronic hypoxia. Meanwhile, the low expression cell model of NEDD4 protein was constructed by the small interference RNA (small interfering RNA, siRNA) technique, and the effect of the expression of NEDD4 protein on the hypoxia tolerance of cardiac myocytes was observed. The purpose of this experiment was to explore the NEDD4 protein. The effect of the regulation of AMPK activity on chronic hypoxic adaptation of the myocardium. 1.: 1. children with congenital heart disease, who were treated in the cardiovascular surgery department of Xinqiao Hospital, Third Military Medical University, January 2013, were divided into cyanotic group and non cyanotic group according to the preoperative blood oxygen saturation. The expression of NEDD4 protein in myocardial tissue was observed by immunohistochemical staining. The total protein in the myocardium was extracted by Ripa lysis method. The expression of Nedd4 protein in the two groups of myocardium and the change of AMPK activation level were detected by Westernblot method, and.2. was cultured under the condition of chronic hypoxia. H9c2 myocardial cells. The total protein of cardiac myocyte was extracted at different time of hypoxia. The expression level of Nedd4 protein and the level of AMPK activation were detected by Westernblot method. At the same time, the siRNA was transfected to the cardiac myocytes with the siRNA of the Nedd4 protein gene of H9c2 cells as the target, and the low expression of Nedd4 protein was established in the chronic hypoxia condition (94%n2,5). %co2,1%o2) after 72h, Westernblot method was used to detect the expression of Nedd4 protein and AMPK activation level. Flow cytometry was used to detect cardiac myocyte apoptosis. Results: 1. children with cyanotic congenital heart disease were significantly lower than non cyanotic group (P0.05) before operation (P0.05), and immunohistochemical detection suggested that Nedd4 protein was mainly in the cytoplasm of cardiac myocytes. Compared with the non cyanotic group, the expression of Nedd4 protein in the myocardium of children with congenital heart disease in cyanosis group was lower (P0.05), and the expression of Nedd4 protein was positively correlated with the blood oxygen saturation of the patients before operation. Compared with those in the non cyanotic group, the AMPK activation level in the cardiac tissue of the cyanotic group was P-A (P-A). Mpk/ampk protein bands with grayscale ratio) significantly enhanced (P0.05).3. in the culture of H9c2 cardiomyocytes under the condition of chronic hypoxia. The Westernblot results showed that the expression level of Nedd4 protein decreased with the prolongation of hypoxia time (P0.05), decreased to the minimum value at hypoxia 24h, and then maintained this low level, and the level of AMPK activation was prolonged with the delay of hypoxia. Long and significant increase (P0.05), at the maximum value of hypoxia 48h, then maintained this high activation level; the expression level of Nedd4 protein expression and the trend of AMPK activation was negatively correlated with.4. flow cytometry: compared with the normal oxygen group, the apoptosis ratio in the anoxic group increased significantly (P0.05), but after the interference of the expression of Nedd4 protein, the hypoxia was caused by hypoxia. The percentage of cardiomyocyte apoptosis decreased (P0.05), and Westernblot results showed that the expression level of Nedd4 protein in the transfected group was significantly lower than that in the negative transfected group (P0.05), and the activation level of AMPK was significantly enhanced (P0.05). Conclusion: 1. compared with the non cyanotic congenital heart disease children, the Nedd4 protein in the myocardium of children with cyanotic congenital heart disease The expression level of AMPK was low, and the activation level of.2. enhanced H9c2 cardiomyocytes under the condition of chronic hypoxia. Compared with the normal oxygen group, the expression of Nedd4 protein in the cardiomyocytes of chronic hypoxia group decreased, the level of AMPK activation enhanced.3. chronic hypoxia to increase the apoptosis of cardiomyocytes, and the AMPK activation level was reduced by siRNA downregulation of the expression of Nedd4 protein in cardiac myocytes. In addition, the percentage of cardiomyocyte apoptosis induced by hypoxia is reduced, which suggests that NEDD4 protein may be involved in chronic hypoxia adaptation by regulating AMPK activity under the condition of chronic hypoxia.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R54

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