本文選題：腺苷酸激活蛋白激酶 + 神經(jīng)前體細(xì)胞表達(dá)的發(fā)育下調(diào)基因4蛋白　； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：目的:慢性缺氧是多種心臟疾病共同的病理生理過程,臨床常見的疾病包括紫紺型先天性心臟病、冠狀動(dòng)脈粥樣硬化性心臟病、代償性心肌肥厚、高原性心臟病等。在慢性缺氧過程中,心肌細(xì)胞面臨嚴(yán)重能量應(yīng)激、氧化應(yīng)激、鈣離子失衡等多種應(yīng)激。應(yīng)激作用導(dǎo)致心肌細(xì)胞自身結(jié)構(gòu)重塑、體液內(nèi)環(huán)境改變等多種適應(yīng)性改變。既往研究證實(shí),這些適應(yīng)性反應(yīng)可促使心肌細(xì)胞耐受多種外界刺激。目前,心肌細(xì)胞慢性缺氧適應(yīng)的確切機(jī)制尚不完全清楚。能量代謝調(diào)節(jié)是心肌細(xì)胞慢性缺氧適應(yīng)的關(guān)鍵環(huán)節(jié)。腺苷酸激活蛋白激酶(AMP-activated Protein Kinase,AMPK)是一種在真核細(xì)胞中廣泛存在的絲氨酸/蘇氨酸蛋白激酶,活化AMPK可調(diào)節(jié)多種信號(hào)通路增加心肌細(xì)胞能量的生成。AMPK被認(rèn)為是心肌細(xì)胞能量代謝調(diào)節(jié)的關(guān)鍵激酶。神經(jīng)前體細(xì)胞表達(dá)的發(fā)育下調(diào)基因4蛋白(Neural precursor cell expressed developmentally down-regulated protein 4,NEDD4)是一種含有HECT結(jié)構(gòu)域的E3泛素連接酶,其結(jié)構(gòu)中的WW結(jié)構(gòu)域可能通過識(shí)別活化AMPK結(jié)構(gòu)中的磷酸化蘇氨酸殘基而參與其活性調(diào)節(jié)。深入研究慢性缺氧時(shí)心肌細(xì)胞中NEDD4蛋白表達(dá)水平和AMPK活化水平的變化以及NEDD4蛋白對(duì)AMPK活性的可能調(diào)節(jié)作用,將有助于我們拓展對(duì)AMPK活性調(diào)節(jié)的認(rèn)識(shí)。本研究以先天性心臟病患兒心肌組織和培養(yǎng)H9c2心肌細(xì)胞作為研究對(duì)象,觀察在慢性缺氧條件下心肌細(xì)胞中NEDD4蛋白表達(dá)水平和AMPK活化水平的變化。同時(shí),利用小干擾RNA(small interfering RNA,siRNA)技術(shù)構(gòu)建NEDD4蛋白低表達(dá)細(xì)胞模型,觀察干擾NEDD4蛋白表達(dá)對(duì)心肌細(xì)胞缺氧耐受的影響。旨在通過上述實(shí)驗(yàn)探討NEDD4蛋白對(duì)AMPK活性的調(diào)節(jié)作用對(duì)心肌慢性缺氧適應(yīng)的影響。方法:1.選取于2013年1月-2014年1月之間在第三軍醫(yī)大學(xué)新橋醫(yī)院心血管外科接受手術(shù)治療的先天性心臟病患兒,根據(jù)患者術(shù)前血氧飽和度分為紫紺組和非紫紺組,取術(shù)中切除的肥厚右室流出道心肌組織作為臨床標(biāo)本,免疫組化染色觀察NEDD4蛋白在心肌組織中的表達(dá)分布情況;ripa裂解法提取心肌標(biāo)本中的總蛋白,采用westernblot法檢測(cè)兩組患兒心肌組織中nedd4蛋白表達(dá)水平及ampk活化水平變化。2.在慢性缺氧條件下培養(yǎng)h9c2心肌細(xì)胞。在不同缺氧時(shí)間段提取心肌細(xì)胞總蛋白,采用westernblot法檢測(cè)nedd4蛋白表達(dá)水平和ampk活化水平變化。同時(shí)將以h9c2心肌細(xì)胞nedd4蛋白基因?yàn)榘悬c(diǎn)的sirna轉(zhuǎn)染至心肌細(xì)胞,建立nedd4蛋白低表達(dá)細(xì)胞模型,在慢性缺氧條件下(94%n2,5%co2,1%o2)培養(yǎng)72h后westernblot法檢測(cè)nedd4蛋白表達(dá)水平與ampk活化水平變化,流式細(xì)胞術(shù)檢測(cè)心肌細(xì)胞凋亡情況。結(jié)果:1.紫紺組先天性心臟病患兒術(shù)前氧飽和度明顯低于非紫紺組(p0.05),免疫組化檢測(cè)提示nedd4蛋白主要在心肌細(xì)胞胞質(zhì)中表達(dá)。與非紫紺組相比,紫紺組先天性心臟病患兒心肌組織中nedd4蛋白表達(dá)水平較低(p0.05),nedd4蛋白表達(dá)水平與患者術(shù)前血氧飽和度呈正相關(guān)。2.westernblot結(jié)果顯示,與非紫紺組患兒相比,紫紺組先天性心臟病患兒心肌組織中ampk活化水平(p-ampk/ampk蛋白條帶灰度比值)顯著增強(qiáng)(p0.05)。3.在慢性缺氧條件下培養(yǎng)h9c2心肌細(xì)胞,westernblot結(jié)果證實(shí),nedd4蛋白表達(dá)水平隨著缺氧時(shí)間的延長(zhǎng)而降低(p0.05),至缺氧24h時(shí)降至最低值,此后維持這一低水平表達(dá);ampk活化水平隨著缺氧時(shí)間的延長(zhǎng)而顯著增加(p0.05),在缺氧48h時(shí)達(dá)最大值,此后維持這一高活化水平;nedd4蛋白表達(dá)水平與ampk活化水平變化趨勢(shì)呈負(fù)相關(guān)。4.流式細(xì)胞術(shù)結(jié)果提示:與常氧組相比,缺氧組心肌細(xì)胞凋亡比例顯著增加(p0.05),但干擾nedd4蛋白表達(dá)后,缺氧所致心肌細(xì)胞凋亡比例降低(p0.05);westernblot結(jié)果提示,與陰性轉(zhuǎn)染組相比,干擾轉(zhuǎn)染組nedd4蛋白表達(dá)水平顯著降低(p0.05),而ampk活化水平顯著增強(qiáng)(p0.05)。結(jié)論:1.與非紫紺型先天性心臟病患兒相比,紫紺型先天性心臟病患兒心肌組織中nedd4蛋白表達(dá)水平較低,ampk活化水平增強(qiáng)。2.在慢性缺氧條件下培養(yǎng)h9c2心肌細(xì)胞,與常氧組相比,慢性缺氧組心肌細(xì)胞中nedd4蛋白表達(dá)水平降低,ampk活化水平增強(qiáng)。3.慢性缺氧增加心肌細(xì)胞凋亡,利用sirna下調(diào)心肌細(xì)胞nedd4蛋白表達(dá)后,ampk活化水平增強(qiáng),缺氧所致的心肌細(xì)胞凋亡比例降低,這提示在慢性缺氧條件下NEDD4蛋白可能通過調(diào)控AMPK活性參與心肌慢性缺氧適應(yīng)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R54

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10 梁俊芳;AMPKα亞基對(duì)小鼠骨骼肌生長(zhǎng)發(fā)育及宰后糖酵解過程的影響[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2013年

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3 王玉兵;HIF-1α、AMPK、E-cadherin在前列腺癌組織中的表達(dá)及意義[D];福建醫(yī)科大學(xué);2015年

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10 閆旭紅;胰島素對(duì)1型糖尿病大鼠睪丸脂聯(lián)素及其受體、AMPK、AKT和eNOS表達(dá)的影響[D];山西醫(yī)科大學(xué);2015年

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