【作者】 金希；

【导师】 厉有名；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 研究背景与目的:非酒精性脂肪性肝病（nonalcoholic fatty liver disease,NAFLD）是指除外酒精和其他明确的肝损因素所致的,以弥漫性肝大泡性脂肪变、伴或不伴炎症为主要特征的临床病理综合征,包括单纯性脂肪肝、脂肪性肝炎、肝纤维化以及肝硬化。近年来,不断有证据提示线粒体功能失常在NAFLD的发生发展中起到重要作用。解偶联蛋白（UCPs）属于线粒体阴离子载体蛋白超家族,定位于线粒体内膜并在多种组织中表达。UCPs通过对氧化磷酸化的解偶联（即质子渗漏）而对线粒体的功能和机体代谢活动产生影响。肝脏是人体最大的代谢器官而其在生理状态下线粒体的质子渗漏占其总耗氧量的20-30%,提示可能存在UCPs在其中起作用。然而迄今为止并未有任何一种UCP在正常肝细胞中表达的报道。肝癌中下调的线粒体载体蛋白（HDMCP）是新近克隆出来并被证明在肝细胞中特异性表达的线粒体载体蛋白,既往研究在细胞株中发现其具有降低线粒体膜电位的作用,因此其可能就是人们长期寻找的肝脏特异性UCP。本研究的目的首先是在酵母菌表达体系中进一步研究HDMCP的解偶联功能;其次研究HDMCP与NAFLD发生发展的关系。研究方法:在C57小鼠肝脏中用Trizol方法提取总mRNA后逆转录为cDNA,再行PCR特异性扩增HDMCP cDNA,后连接至PMDT₁₈载体,转化大肠杆菌后过夜培养,提取质粒后送测序。将测序证实无突变的PMDT₁₈-HDMCP质粒再PCR扩增并与pYES2载体连接,同样转化大肠杆菌后质粒测序。将测序正确的pYES2-HDMCP质粒转化酵母菌,经过1%的半乳糖诱导表达后,提取线粒体并立即在30℃下进行呼吸功能测定,主要指标包括解偶联率、线粒体呼吸控制及GDP抑制率。随后,本课题通过高脂饮食和油酸、棕榈酸混合物培养成功构建NAFLD大鼠及脂质沉积细胞模型,并使用western blot、RNA干扰等方法研究HDMC在脂质沉积中的作用。最后,本课题还采用分光光度法和萤火虫荧光素酶法分别测定细胞模型中过氧化氢和ATP含量,以研究HDMCP调控脂质水平的下游通路。本课题比较多地采用生物信息学的方法,如对HDMCP与UCP1-5的序列比对采用clustalW程序,进化分析主要采用Mega 4软件包中的邻近法进行构树。在统计学上,每项实验至少进行三次,计量资料采用平均数±标准误来表示。非配对的两组间比较采用student’s t检验（对于非正态分布的两组间数据比较采用Mann-Whitney法）,两变量间相互关系的分析采用直线相关分析进行。所有统计数据均由专人录入并在SPSS 11.0中进行分析,p＜0.05为有显著性差异。结果:通过序列比对发现HDMCP与UCP1-5有约40%的序列相似度,且表现出线粒体载体蛋白的基本特征。进一步通过进化分析,发现HDMCPs显著的与UCP4,UCP5以及无脊椎动物中的UCPs序列更相近,提示进化上其可能更加原始。Westen blot显示HDMCP在分离的酵母线粒体中表达。由于缺乏OXO和UCP1的特异性抗体,本课题采用实时定量RT-PCR证实了二者mRNA在各自转染的酵母线粒体上的表达（OXO:0.73±0.07;UCP1 0.81±0.12）。在提取线粒体并测定解偶联率时,本课题发现HDMCP具有一定GDP非敏感性的解偶联功能,与OXO和pyes2阴性对照及UCP1阳性对照相比,差异具有统计学意义（p＜0.05）。与对照相比,肝指数、血清胆固醇与甘油三酯、肝脏甘油三酯含量以及HOMA-IR指数在NAFLD均有显著性增高。此外,western blot显示HDMCP在NAFLD模型组中显著增高。而细胞脂质含量及甘油三酯水平在模型组也比对照组有2-7倍的增高。随着高脂培养时间的延长,HDMCP水平逐渐上升。通过RNA干扰,L02与HepG2细胞中HDMCP的表达分别下调了60%与62%,伴细胞脂质含量及甘油三酯的显著增高,而control-shRNA则无显著作用。此外,NAFLD动物与细胞模型中的ATP含量以及细胞模型中的H₂O₂（ROS的代表物）水平比对照均呈显著下降,然而这种作用部分被HDMCP-shRNA通过下调HDMCP水平所抑制。同时ATP水平与其相应的HDMCP水平呈负相关（r=-0.93,L02细胞,p＜0.05;r=-0.91 HepG2细胞,p＜0.05）。结论:1)通过在酵母表达系统中表达HDMCP,本课题验证了其具有GDP非敏感性的解偶联功能,支持“HDMCP可能就是研究人员长期寻找的肝脏特异性的解偶联蛋白”这一假说,为全面理解生理状态下肝脏线粒体能量代谢提供理论支持。2)本课题首次发现并阐明HDMCP在NAFLD大鼠模型及单纯脂变模型中表达上升,通过进一步研究发现HDMCP具有部分调节NAFLD细胞模型中脂质沉积的作用,而且该调控可能通过降低ATP与H₂O₂的产生起作用,可能是机体面对脂肪过剩情况下的适应性反应。因此,HDMCP可能成为今后NAFLD治疗干预的新靶点。3)由于H₂O₂是脂质过氧化的重要产物,因此,本课题的研究也提示HDMCP可能在NAFLD从单纯性脂变向NASH的进展中起到重要作用。更多还原

【Abstract】 Backgrond/aims:Nonalcoholic fatty liver disease（NAFLD） represents a common clinicopathologic condition characterized by lipid deposition in hepatocytes of liver parenchyma without alcohol consumption in amounts considered to be harmful to the liver,ranging from simple steatosis to steatohepatitis,fibrosis,cirrhosis and hepatocellular carcinoma. Nowadays,there are accumulating evidences that mitochondrial dysfunction plays a pivotal role in NAFLD.Uncoupling proteins（UCPs） belong to the superfamily of mitochondrial anion-carrier proteins,which are located on the mitochondrial inner membrane and are identified in various tissues.UCPs uncouple the mitochondrial respiration from ATP synthesis by dissipating the transmembrane proton gradient to further influence mitochondrial function and metabolic process.Therefore,UCPs might be a key cluster of proteins involved in mitochondrial dysfunction.Liver is the largest metabolic organ in human body and mitochondrial proton leak accounts for 20-30%of the oxygen consumption of isolated resting hepatocytes.Due to the uncoupling character of UCPs,it is plausible that they might participate in hepatic mitochondrial proton leak and certain dysregulated metabolic pathways.Nevertheless, there are currently no UCPs（UCP 1 to 5） detected in normal hepatocytes. Hepatocellular carcinoma down-regulated mitochondrial carrier protein（HDMCP） was first cloned in the year of 2004 and proved to be exclusively expressed in liver.This protein bears all the hallmark features of the mitochondrial anion-carrier proteins and is significantly down-regulated during the development of hepatocellular carcinoma. However,whether it does have an uncoupling activity needs to be examined by more experimental systems.Therefore,our aim of this study is to explore the uncoupling activity of HDMCP in a yeast expression system and its function in NAFLD.Methods:CDNAs of UCP1,OXO and HDMCP were obtained using routine RT-PCR amplification from mice livers and then sequentially cloned into PMDT₁₈ and pYES2 expression vector.Thereafter,Saccharomyces cerevisae was transformed with purified pYES2 vectors containing UCP1,OXO,HDMCP and empty vector and further grown in SC-ura liquid medium.After approximately 48h growth,mitochondria were isolated from the yeast and the uncoupling activity was measured.NAFLD cell model was established by exposing L02 and HepG2 cells at 80%confluency to HFFA,a mixture of oleate（OA） and palmitate（PA）,at the final ratio of 2:1 and concentration of 1mM for serial time spots（24 h,36h,48h,60h and 72h）.Nile Red（1 mg/ml in PBS） was used to display the cellular lipid content.Cellular protein content was assessed by the lowry method and TG level was measured using the method of GPO/POD enzymatic reaction. For NAFLD animal model,a total of 24 twelve-week-old SDrats weighing 158g-172g were randomly divided into NAFLD（n=12） and control group（n=12）.Control group was given basic diet while NAFLD group was given fat-rich diet.The successful establishment of both two models was confirmed by pathologic（Nile red and H-E staining） and biochemical changes（TG,Tch,fasting glucose,fasting insulin,et al）.Routine real time RT-PCR and western blot were used to detect HDMCP mRNA and protein levels.RNA interference was used to knockdown HDMCP level by adding HDMCP-shRNA into cell models using Lipofectamine 2000.Mitochondrial ATP was extracted and measured using a luciferin-luciferase bioluminescent assay.The rate of mitochondrial H₂O₂ production was determined according to the manufacturer’s instructions.Each experiment was performed in at least triplicate and data were expressed as means±SE（standard error）.Unpaired student’s t-test for normal distributed numerical variance and Mann-Whitney for skewed data were executed by SPSS 11.0.Linear correlation was used to explore the association between two variables.The differences were considered statistically significant at p＜0.05.Results:Firstly,we successfully expressed HDMCP in yeast mitochondria and found a significant GDP insensitive uncoupling activity of HDMCP.Secondly,on the basis of successfully established animal and cell NAFLD models,we found a significantly increased expression of HDMCP in both two models,where the increment of HDMCP in cell model was correlated with culture time and steatosis was aggravated when HDMCP level was knocked down by RNA interference.Finally,we found that the effect of HDMCP in steatosis alleviation might function through promoting ATP depletion and decreasing H₂O₂ production,as these two markers were both significantly decreased in L02 and HepG2 cells with high expression of HDMCP.Furthermore, down-regulation of HDMCP could significantly antagonize these changes.Conclusion:This study adds supportive data to the hypothesis that HDMCP might be a long postulated liver specific uncoupling protein and broaden our understanding of the pathogenesis of NAFLD.More importantly,HDMCP might become a novel drug target for its property in alleviating hepatic steatosis.更多还原