本文關(guān)鍵詞：3-巰基丙酮酸轉(zhuǎn)硫酶在非酒精性脂肪性肝病中的作用及其分子機制研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

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【摘要】：背景和目的:肝臟中脂肪酸的過度沉積會通過脂毒性導(dǎo)致非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)的發(fā)生發(fā)展,但其中涉及的分子機制尚不明確。胱硫醚-β-合酶(cystathionine-β-synthase,CBS)和胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)是體內(nèi)內(nèi)源性硫化氫(hydrogensulfide,H2S)的主要產(chǎn)生酶,已被發(fā)現(xiàn)受到脂肪酸的調(diào)控并參與NAFLD發(fā)病機制。本研究旨在探究另一內(nèi)源性H2S產(chǎn)生酶,3-巰基丙酮酸轉(zhuǎn)移硫酶(3-mercaptopyruvate sulfurtransferase,MPST)在 NAFLD 發(fā)生發(fā)展中的作用及機制。材料和方法:采用游離脂肪酸(free fatty acid,FFA)(油酸:棕櫚酸為2:1)混合培養(yǎng)液刺激人正常肝細(xì)胞L02細(xì)胞,及高脂飲食(high fat diet,HFD)喂養(yǎng)C57BL/6小鼠分別建立NAFLD細(xì)胞及動物模型,用Western Blot法檢測MPST在NAFLD模型中的表達水平。采用免疫組化法檢測MPST在NAFLD臨床患者肝臟標(biāo)本中的蛋白表達水平。采用重組腺病毒介導(dǎo)shRNA結(jié)合尾靜脈高壓注射技術(shù)對小鼠體內(nèi)肝臟MPST的表達進行干預(yù),并在借助轉(zhuǎn)錄激活樣效應(yīng)因子核酸酶(transcription activator-like effector nuclease,TALEN)技術(shù)構(gòu)建的 C57BL/6 為背景的 MPST 基因敲除雜合子小鼠中進一步觀察MPST對肝臟脂肪變性的影響作用。在FFA刺激的L02細(xì)胞中,分別運用siRNA和轉(zhuǎn)染MPST過表達質(zhì)粒以干預(yù)MPST表達,觀察MPST對肝細(xì)胞脂肪變性的影響與作用。采用HE及油紅O染色觀察肝臟和肝細(xì)胞脂變程度,通過測定肝甘油三酯(TG)、總膽固醇(TC)含量及FFA水平,聯(lián)系H2S水平變化,MDA及SOD活性等指標(biāo)變化研究MPST對肝臟脂肪變性及氧化應(yīng)激的影響。通過外源性H2S供體的應(yīng)用,檢測CSE及SREBP-1通路、JNK磷酸化水平變化,免疫共沉淀(Co-Imnunoprecipitation,CoIP)技術(shù)探究潛在的分子機制。結(jié)果:FFA的刺激誘導(dǎo)MPST在人脂變肝細(xì)胞中表達上調(diào),并部分依賴于NF-1κB/p65。在HFD誘導(dǎo)的小鼠NAFLD模型及臨床NAFLD患者肝臟中,MPST蛋白表達水平顯著升高。應(yīng)用MPST-shRNA重組腺病毒尾靜脈高壓注射介導(dǎo)小鼠肝臟MPST表達部分敲低,和使用MPST基因敲除雜合子小鼠介導(dǎo)遺傳性的MPST表達部分缺失,均能顯著改善HFD誘導(dǎo)的肝臟脂肪變性程度。和體內(nèi)水平一致地,在L02細(xì)胞中抑制MPST表達后,FFA誘導(dǎo)的肝細(xì)胞脂變顯著改善,而過表達MPST后則脂變加劇。有趣的是,部分敲低MPST會顯著上調(diào)H2S水平,相反地,過表達MPST則顯著下調(diào)H2S水平。CoIP實驗揭示MPST可以通過直接蛋白-蛋白相互作用而負(fù)調(diào)控CSE——肝臟中H2S產(chǎn)生主要酶。且進一步的機制研究發(fā)現(xiàn),H2S在MPST對脂肪肝的調(diào)節(jié)過程中起重要介導(dǎo)作用,與脂肪合成關(guān)鍵蛋白SREBP-1相關(guān)通路抑制,JNK磷酸化減弱及肝臟氧化應(yīng)激改善有關(guān)。結(jié)論:FFA介導(dǎo)MPST表達顯著上調(diào),MPST通過CSE依賴途徑調(diào)節(jié)H2S水平,在NAFLD發(fā)生發(fā)展中起到重要作用。這為基于MPST為潛在靶點的NAFLD新藥研發(fā)提供了新的理論依據(jù),部分抑制MPST有望成為治療NAFLD新策略。
[Abstract]:Background and purpose: excessive deposition of fatty acids in the liver can lead to the occurrence and development of nonalcoholic fatty liver disease (NAFLD) through lipotoxicity. However, the molecular mechanism involved is not clear. Cystin cystathionine- -synthase (CBS) and cystathionine- -lyase -lyase (CSE) are the main enzymes producing endogenous hydrogen sulfide (HYDROGENSULFIDE, H2S). They have been found to be regulated by fatty acids and participate in the pathogenesis of NAFLD. The purpose of this study is to explore the role and mechanism of another endogenous H2S producing enzyme, 3- thiol pyruvate transferase (3-mercaptopyruvate sulfurtransferase (MPST)) in the occurrence and development of NAFLD. Materials and methods: using free fatty acids (free fatty, acid, FFA) (oleic acid palmitic acid 2:1) mixed medium stimulation of normal human liver cells L02 cells, and high fat diet (high fat, diet, HFD) NAFLD cells and animal models were established by feeding C57BL/6 mice, the expression level of Western detected by Blot MPST in the NAFLD model. The protein expression level of MPST in the liver specimens of patients with NAFLD was detected by immunohistochemistry. Were treated with recombinant adenovirus mediated shRNA combined with tail vein injection of mouse liver MPST expression, and with the help of transcription activator likeeffector nuclease (transcription activator-like effector nuclease TALEN) technology to build C57BL/6 for the background of MPST gene knockout further observe the effect of MPST on hepatic steatosis in heterozygous effect in mice. In the L02 cells stimulated by FFA, siRNA and MPST overexpression plasmids were used to interfere with the expression of MPST, and the effect of MPST on the fatty degeneration of hepatocytes was observed. HE and oil red O staining were used to observe the degree of liver and hepatocyte lipidosis. The effects of MPST on hepatic steatosis and oxidative stress were studied by measuring the contents of hepatic triglyceride (TG), total cholesterol (TC) and FFA level, and the changes of H2S level, MDA and SOD activity. Through the application of exogenous H2S donors, we detected the changes of CSE and SREBP-1 pathway and JNK phosphorylation level, and explored the potential molecular mechanism of Co-Imnunoprecipitation (CoIP) technology. Results: the expression of MPST was up-regulated in human lipoprotein hepatocytes by FFA stimulation, and partly dependent on NF-1 kappa B/p65. The expression of MPST protein was significantly increased in the HFD induced mouse NAFLD model and the liver of the clinical NAFLD patients. MPST-shRNA recombinant adenovirus mediated tail vein high pressure injection mediated partial knockdown of MPST expression in mice liver, and partial deletion of MPST expression mediated by MPST knockout heterozygous mice can significantly improve the degree of HFD induced hepatic steatosis. After the expression of MPST in L02 cells was consistent with the level of the body, the liver cell lipid induced by FFA significantly improved, and the fat increased after the overexpression of MPST. Interestingly, partial knockout MPST significantly up-regulated the H2S level, and on the contrary, overexpression of MPST significantly lowered the level of H2S. The CoIP experiment revealed that MPST can negatively regulate CSE by direct protein protein interaction - the main enzyme produced by H2S in the liver. Further mechanism studies showed that H2S plays an important mediating role in the regulation of MPST on fatty liver, and is related to inhibition of SREBP-1 related pathway, JNK phosphorylation and liver oxidative stress. Conclusion: FFA mediated MPST expression is significantly up-regulated, and MPST regulates the level of H2S through CSE dependent pathway, which plays an important role in the development of NAFLD. This provides a new theoretical basis for the research and development of new NAFLD drugs based on MPST as a potential target, and partial inhibition of MPST is expected to be a new strategy for the treatment of NAFLD.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R575.5

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相關(guān)期刊論文 前1條

1 Sarathi Mani;Hongzhu Li;Guangdong Yang;Lingyun Wu;Rui Wang;;CSE/H_2S通路缺失影響小鼠肝臟膽固醇和脂肪酸代謝(英文)[J];Science Bulletin;2015年03期

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本文編號：1342264