【作者】 晁帆；

【导师】 何凤田；

【作者基本信息】 第三军医大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 研究背景:动脉粥样硬化(atherosclerosis,AS)是严重威胁人类健康的心血管疾病。血浆胆固醇水平与动脉粥样硬化斑块的形成密切相关。B类清道夫受体I(scavenger receptor class B type I,SR-BI)是高密度脂蛋白胆固醇(high density lipoprotein-cholesterol,HDL-C)的高亲和力受体,主要介导血浆胆固醇逆向转运回肝脏,从而有利于防止AS的发生和发展。法尼酯X受体(farnesoid X receptor,FXR)是核受体超家族成员,通过调节诸多靶基因而在调控胆固醇、脂类和葡萄糖的代谢过程中发挥着极其重要的作用,FXR功能丧失与AS密切相关。然而,关于FXR功能丧失到底是促进还是抑制AS的发展,目前尚难以定论。由于FXR和SR-BI均高表达于肝脏,且二者均与AS密切相关,那么SR-BI是否是FXR的一个新靶基因,其表达是否可受到FXR的调节?弄清该问题,将为进一步阐明二者在AS发生发展中的作用及为寻找防治AS的新靶点提供新的科学依据。研究目的:探讨FXR对SR-BI表达的影响及可能机制。研究方法:选取人内皮细胞Eahy926、胎肝细胞L02和肝癌细胞HepG2为细胞模型。经FXR激动剂GW4064、androsterone和CDCA分别处理24h后,RT-PCR和Real-time PCR法检测SR-BI mRNA表达的变化;western blot法检测SR-BI蛋白表达的差异。在线预测SR-BI基因5’侧翼启动子区域(-1200～-59bp)可能存在的FXR结合位点;荧光素酶报告基因检测FXR对SR-BI启动子的调控;采用电泳迁移率变动实验,观察FXR对SR-BI启动子区域的直接结合作用。利用RT-PCR和Real-time PCR法检测PPARγ的表达;经PPARγ拮抗剂GW9662和FXR激动剂GW4064先后作用,采用RT-PCR、Real-time PCR和western blot法检测SR-BI表达的变化;并进一步用荧光素酶报告基因检测GW9662对SR-BI启动子的调控。经PPARγ激动剂troglitazone处理HepG2细胞,RT-PCR、Real-time PCR和western blot法检测SR-BI mRNA和蛋白表达的变化。在体动物喂食含CDCA的食物,RT-PCR、Real-time PCR和western blot法检测其SR-BI表达的变化。研究结果:(1)在FXR激动剂处理的细胞系中,发现FXR呈配体剂量依赖性的上调SR-BI mRNA和蛋白表达;(2)在HepG2细胞中,FXR可上调SR-BI启动子的活性,并可直接结合于SR-BI启动子区域(-1200/-59bp)DR8元件,上调SR-BI的表达;(3)在FXR激动剂处理的细胞系中,发现FXR可在转录水平上调PPARγ的表达,PPARγ拮抗剂可抑制FXR激动剂对SR-BI的诱导表达作用;(4)在HepG2细胞中,PPARγ激动剂可上调SR-BI mRNA和蛋白表达;(5)小鼠喂食含CDCA的食物,FXR可上调SR-BI的表达。结论:(1)体外研究显示,FXR可通过结合于SR-BI启动子区的DR8位点直接上调SR-BI的表达;(2)FXR也可经上调PPARγ而间接诱导SR-BI的表达;(3)在体动物实验显示,FXR可在小鼠体内上调SR-BI的表达。以上有关研究,为深入认识FXR和SR-BI在胆固醇代谢以及AS等相关疾病中的作用具有重要意义,二者有可能成为防治AS等相关疾病的新靶点。更多还原

【Abstract】 Background: Plasma concentrations of high-density lipoprotein (HDL) cholesterol are well known to be inversely related to the incidence of atherosclerosis and coronary heart disease. Scavenger receptor type B class I as HDL receptor plays an important role in reverse cholesterol transport, protected against AS. The nuclear farnesoid X receptor plays an important role in in glucose metabolism, cholesterol metabolism, TG metabolism. And there is a closely link with the FXR lacking and AS. Whether depletion of FXR induce or inhibit AS, remains controversial. FXR and SR-BI both are highly expressed in liver, then whether FXR could regulate SR-BI to inhibit AS? The study on the effect of FXR on SR-BI will provide useful information with regarding to the physiological and pathological roles FXR and SR-BI play in cholesterol metabolism, and supply a potential drug target.Objective: the present study investigated the effect of FXR on SR-BI expression and the mechanism by which FXR enhances the expression of the SR-BI.Methods: endothelial cells, hepatocyte and hepatoma cell lines were employed for investigation. Three cell lines were stimulated with the given different concentrations of FXR agonists, respectively. Using RT-PCR, Real-time PCR and western blot detected the SR-BI expression levels. Bioinformatics analysis (http://www.nubiscan.unibas.ch/) indicated that there is a potential FXR binding site (DR8) in the SR-BI promoter region (-1200/-59bp). Luciferase reporter assay detected the SR-BI promoter activity with FXR. And EMSA was performed to detect FXR protein associated with SR-BI. RT-PCR and Real-time PCR was performed to detected PPARγexpression. The combination of GW9662 with GW4064 treat HepG2, RT-PCR, Real-time PCR and western blot were performed to detect the SR-BI expression. Luciferase reporter assay detected the SR-BI promoter activity with GW9662. With RT-PCR, Real-time PCR and western blot, we detected SR-BI expression treatment with PPARγagonist troglitazone. In vivo study, upon treatment of FXR agonsit CDCA, SR-BI mRNA and protein were analysed with RT-PCR, Real-time PCR and Western Blot.Results: (1) treatment with FXR agonists, FXR could up-regulate SR-BI expression in concentration-dependent manner;(2) in HepG2, FXR enhanced SR-BI promoter activity, and up-regulate SR-BI expression through FXR binding site(DR8) in SR-BI promoter region;(3) FXR induced PPARγat transcription level, PPARγantagonist GW9662 repressed the SR-BI promoter activity which was stimulated by FXR;(4) in HepG2, PPARγcould up-regulate SR-BI expression;(5) in C57BL/6 mice FXR up-regulated the expression of SR-BI.Conclusion: (1) we identified SR-BI was a target gene of FXR, which could be directly up-regulated by FXR. There is a putative FXR binding site (DR8) within the SR-BI promoter region;(2) we also investigated the indirect mechanism that FXR mediates SR-BI through PPARγ;(3) in vivo study, we demonstrated that FXR could up-regulate SR-BI in mice.The study on the effect of FXR on SR-BI will provide useful information with regarding to the physiological and pathological roles FXR plays in cholesterol metabolism, and suggests a new therapy.更多还原