本文選題：腺苷酸激活蛋白激酶 + 神經(jīng)前體細胞表達的發(fā)育下調基因4蛋白�。� 參考：《第三軍醫(yī)大學》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,心肌細胞面臨嚴重能量應激、氧化應激、鈣離子失衡等多種應激。應激作用導致心肌細胞自身結構重塑、體液內環(huán)境改變等多種適應性改變。既往研究證實,這些適應性反應可促使心肌細胞耐受多種外界刺激。目前,心肌細胞慢性缺氧適應的確切機制尚不完全清楚。能量代謝調節(jié)是心肌細胞慢性缺氧適應的關鍵環(huán)節(jié)。腺苷酸激活蛋白激酶(AMP-activated Protein Kinase,AMPK)是一種在真核細胞中廣泛存在的絲氨酸/蘇氨酸蛋白激酶,活化AMPK可調節(jié)多種信號通路增加心肌細胞能量的生成。AMPK被認為是心肌細胞能量代謝調節(jié)的關鍵激酶。神經(jīng)前體細胞表達的發(fā)育下調基因4蛋白(Neural precursor cell expressed developmentally down-regulated protein 4,NEDD4)是一種含有HECT結構域的E3泛素連接酶,其結構中的WW結構域可能通過識別活化AMPK結構中的磷酸化蘇氨酸殘基而參與其活性調節(jié)。深入研究慢性缺氧時心肌細胞中NEDD4蛋白表達水平和AMPK活化水平的變化以及NEDD4蛋白對AMPK活性的可能調節(jié)作用,將有助于我們拓展對AMPK活性調節(jié)的認識。本研究以先天性心臟病患兒心肌組織和培養(yǎng)H9c2心肌細胞作為研究對象,觀察在慢性缺氧條件下心肌細胞中NEDD4蛋白表達水平和AMPK活化水平的變化。同時,利用小干擾RNA(small interfering RNA,siRNA)技術構建NEDD4蛋白低表達細胞模型,觀察干擾NEDD4蛋白表達對心肌細胞缺氧耐受的影響。旨在通過上述實驗探討NEDD4蛋白對AMPK活性的調節(jié)作用對心肌慢性缺氧適應的影響。方法:1.選取于2013年1月-2014年1月之間在第三軍醫(yī)大學新橋醫(yī)院心血管外科接受手術治療的先天性心臟病患兒,根據(jù)患者術前血氧飽和度分為紫紺組和非紫紺組,取術中切除的肥厚右室流出道心肌組織作為臨床標本,免疫組化染色觀察NEDD4蛋白在心肌組織中的表達分布情況;ripa裂解法提取心肌標本中的總蛋白,采用westernblot法檢測兩組患兒心肌組織中nedd4蛋白表達水平及ampk活化水平變化。2.在慢性缺氧條件下培養(yǎng)h9c2心肌細胞。在不同缺氧時間段提取心肌細胞總蛋白,采用westernblot法檢測nedd4蛋白表達水平和ampk活化水平變化。同時將以h9c2心肌細胞nedd4蛋白基因為靶點的sirna轉染至心肌細胞,建立nedd4蛋白低表達細胞模型,在慢性缺氧條件下(94%n2,5%co2,1%o2)培養(yǎng)72h后westernblot法檢測nedd4蛋白表達水平與ampk活化水平變化,流式細胞術檢測心肌細胞凋亡情況。結果:1.紫紺組先天性心臟病患兒術前氧飽和度明顯低于非紫紺組(p0.05),免疫組化檢測提示nedd4蛋白主要在心肌細胞胞質中表達。與非紫紺組相比,紫紺組先天性心臟病患兒心肌組織中nedd4蛋白表達水平較低(p0.05),nedd4蛋白表達水平與患者術前血氧飽和度呈正相關。2.westernblot結果顯示,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(p0.05)。3.在慢性缺氧條件下培養(yǎng)h9c2心肌細胞,westernblot結果證實,nedd4蛋白表達水平隨著缺氧時間的延長而降低(p0.05),至缺氧24h時降至最低值,此后維持這一低水平表達;ampk活化水平隨著缺氧時間的延長而顯著增加(p0.05),在缺氧48h時達最大值,此后維持這一高活化水平;nedd4蛋白表達水平與ampk活化水平變化趨勢呈負相關。4.流式細胞術結果提示:與常氧組相比,缺氧組心肌細胞凋亡比例顯著增加(p0.05),但干擾nedd4蛋白表達后,缺氧所致心肌細胞凋亡比例降低(p0.05);westernblot結果提示,與陰性轉染組相比,干擾轉染組nedd4蛋白表達水平顯著降低(p0.05),而ampk活化水平顯著增強(p0.05)。結論: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蛋白表達水平較低,ampk活化水平增強。2.在慢性缺氧條件下培養(yǎng)h9c2心肌細胞,與常氧組相比,慢性缺氧組心肌細胞中nedd4蛋白表達水平降低,ampk活化水平增強。3.慢性缺氧增加心肌細胞凋亡,利用sirna下調心肌細胞nedd4蛋白表達后,ampk活化水平增強,缺氧所致的心肌細胞凋亡比例降低,這提示在慢性缺氧條件下NEDD4蛋白可能通過調控AMPK活性參與心肌慢性缺氧適應。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R54

【相似文獻】

相關期刊論文 前10條

1 李娟娟;凌文華;;AMPK與肥胖[J];國際內科學雜志;2007年11期

2 劉文倩;艾華;;AMPK與肥胖和減肥關系研究進展[J];中國運動醫(yī)學雜志;2008年06期

3 蔡明春,黃慶愿,高鈺琪;AMPK與能量代謝[J];重慶醫(yī)學;2005年01期

4 葛斌;謝梅林;顧振綸;周文軒;郭次儀;;AMPK作為治療2型糖尿病新靶點的研究進展[J];中國藥理學通報;2008年05期

5 徐靜;劉毅;完強;賈振華;王榮;;高糖環(huán)境對大鼠腎小球系膜細胞AMPK表達及活性的影響[J];山東大學學報(醫(yī)學版);2009年05期

6 宮克城;丁樹哲;;AMPK與2型糖尿病的關系及其在運動介導下的研究[J];遼寧體育科技;2009年03期

7 羅招凡;李芳萍;丁鶴林;程樺;;AMPKα2基因克隆及其野生型和突變型真核表達載體的構建[J];中國組織工程研究與臨床康復;2009年28期

8 丁曉潔;王佑民;王麗萍;;高脂飲食對大鼠肝臟組織AMPK表達及其活性的影響[J];安徽醫(yī)科大學學報;2009年06期

9 程媛;王佑民;丁曉潔;;肥胖大鼠骨骼肌AMPK表達及其與糖脂代謝的關系[J];安徽醫(yī)科大學學報;2010年02期

10 黃德強;羅凌玉;王麗麗;羅時文;呂農華;羅志軍;;AMPK在胰島素信號轉導通路中的作用[J];中國細胞生物學學報;2011年11期

相關會議論文 前10條

1 ;Co-commitment and Interplay between Akt and AMPK in the Regulation of Endothelial NO Synthase Phosphorylation by Reactive Oxygen Species[A];第九屆全國心血管藥理學術會議論文集[C];2007年

2 Paul M Vanhoutte;;SIRT1 and AMPK:the Seesaw Effect in Regulating Endothelial Sene-scence[A];第八屆海峽兩岸心血管科學研討會論文集[C];2011年

3 李瑾;朱海波;;新結構類型調血脂化合物與靶蛋白AMPK分子間相互作用初步解析[A];全國第十二屆生化與分子藥理學學術會議論文集[C];2011年

4 宋海燕;李強;孫玉倩;張巾超;鄔艷慧;;AMPK結合蛋白的篩選及其與2型糖尿病的關系研究[A];2008內分泌代謝性疾病系列研討會暨中青年英文論壇論文匯編[C];2008年

5 ;AMPK mediated an apoptotic response to combined effect of hypoxia stress and ER stress[A];2012全國發(fā)育生物學大會摘要集[C];2012年

6 ZHANG Jian-wei;MA Xiao-wei;DENG Rui-fen;DING Shan;GU Nan;GUO Xiao-hui;;Genetic variability in AMPKαl gene may have synergetic effect with smoking on risk of coronary artery disease in chinese type 2 diabetics[A];中華醫(yī)學會糖尿病學分會第十六次全國學術會議論文集[C];2012年

7 姚遠;周京軍;裴建明;;AMPK介導無鈣預處理心肌保護作用[A];中國生理學會第九屆全國青年生理學工作者學術會議論文摘要[C];2011年

8 季樂樂;Haifeng Zhang;Feng Gao;;A novel mechanism of preconditioning:Attenuating reperfusion injury through enhanced myocardial glucose uptake via insulin-stimulated Akt and AMPK activation[A];中國生理學會第十屆全國青年生理學工作者學術會議論文摘要[C];2013年

9 WU Qiao;;The orphan nuclear receptor Nur77 regulates AMPK activity through LKB1 subcellular localization in glucose metabolism[A];細胞—生命的基礎——中國細胞生物學學會2013年全國學術大會·武漢論文摘要集[C];2013年

10 Jia-Wei Wu;;Conserved elements in allosteric regulation of AMPK[A];中國生物化學與分子生物學會第十一次會員代表大會暨2014年全國學術會議論文集——專題報告二[C];2014年

相關重要報紙文章 前2條

1 黃敏;精力充沛基因決定?[N];新華每日電訊;2011年

2 實習生 程鳳;不愛鍛煉可能與基因缺失有關[N];科技日報;2011年

相關博士學位論文 前10條

1 陳雷;AMP激活的蛋白質激酶（AMPK）調控機制的研究[D];清華大學;2010年

2 張秀娟;TSH調節(jié)肝臟HMG-CoA還原酶磷酸化修飾的研究[D];山東大學;2014年

3 趙順玉;消積飲聯(lián)合CIK通過AMPKα/Sp1/EZH2/DNMT1相關通路抗肺癌生長的作用機制[D];廣州中醫(yī)藥大學;2015年

4 王紅亮;AMPK-α在鹵蟲胚胎發(fā)育過程中對細胞有絲分裂調控的研究[D];浙江大學;2015年

5 周錫紅;三甲基甘氨酸通過AMPK途徑影響脂肪沉積的研究[D];浙江大學;2015年

6 朱莉;電鏡單顆粒技術研究全長AMPK蛋白的構架及變構效應[D];蘭州大學;2011年

7 符慶瑛;大腸桿菌雙雜交篩選AMPKα2相互作用蛋白[D];第三軍醫(yī)大學;2007年

8 鄧虎平;燙傷大鼠骨骼肌AMPK的活化對蛋白分解的作用及其信號轉導機制[D];中國人民解放軍軍醫(yī)進修學院;2010年

9 余冰;AMPK對應激狀態(tài)下仔豬脂質代謝的調節(jié)作用[D];四川農業(yè)大學;2003年

10 梁俊芳;AMPKα亞基對小鼠骨骼肌生長發(fā)育及宰后糖酵解過程的影響[D];內蒙古農業(yè)大學;2013年

相關碩士學位論文 前10條

1 柯志強;AMPK信號通路在白藜蘆醇改善高糖誘導乳鼠心肌細胞損傷中的作用[D];湖北科技學院;2015年

2 肖瑤;AMPK對低氧誘導血管生成作用的研究[D];湖北科技學院;2015年

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

4 魏蘇玉;乙醇對H4-ⅡE細胞脂質代謝及AMPK表達的影響[D];延邊大學;2015年

5 陳婷;肌肉特異敲除AMPKα2對小鼠脂代謝的影響[D];西北農林科技大學;2015年

6 張二東;銅離子及模擬太空環(huán)境通過ROS/AMPK信號誘導人B淋巴母細胞凋亡[D];蘭州大學;2015年

7 徐英秀;硫化氫通過AMPK激活調控腦缺血后小膠質細胞的極化狀態(tài)[D];蘇州大學;2015年

8 黃艷;AMPK和SIRT-1參與定時高脂飲食對小鼠肝臟生物鐘基因的影響研究[D];蘇州大學;2015年

9 楊霞;AMPK參與線粒體通路介導的氟致H9c2心肌細胞凋亡機制的研究[D];山西醫(yī)科大學;2015年

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

，

本文編號：1924359