转录因子Sp1对胆固醇逆转运关键基因调控机制的研究

发布时间：2018-01-18 09:36

本文关键词：转录因子Sp1对胆固醇逆转运关键基因调控机制的研究　出处：《北京协和医学院》2014年博士论文　论文类型：学位论文

【摘要】：B族I型清道夫受体(Scavenger receptor class B type Ⅰ, SR-BI)是胆固醇逆转运过程中关键蛋白之一,它通过选择性摄取血浆中的HDL胆固醇(HDL-C)至肝细胞中,调节体内HDL-C水平。因而SR-BI的表达调控对于调节血脂水平,降低心血管疾病的风险具有重要意义。本研究在第一部分发现了高脂饮食的ApoE-/-小鼠肝组织中,以及低密度脂蛋白LDL处理的肝细胞HepG2中,SR-BI表达均明显增加。为了研究SR-BI表达调控的机制,我们首先采用SR-BI基因启动子荧光素酶报告基因和RNAi等方法,发现了转录因子Spl在这一调控过程中起到关键作用,当用Sp1-siRNA抑制细胞Spl表达后,LDL便不能激活SR-BI的表达。但在HepG2细胞中过表达Spl对SR-BI基因并无明显激活作用,而抑制Spl的活性后SR-BI的表达水平明显降低。一系列的免疫共沉淀实验表明,LDL激活SR-BI表达时,SR-BI启动子区域Spl与组蛋白乙酰化酶p300和组蛋白去乙酰化酶HDAC1构成的蛋白质复合物发生了改变,Sp1募集了更多的p300且同时与HDA C1分离,导致组蛋白乙酰化水平增加,从而激活基因转录。之后进一步考察了Spl蛋白质复合物发生改变的原因,发现在LDL的作用下,Spl的蛋白质翻译后修饰发生了变化,其磷酸化水平明显增加。通过对HepG2细胞在LDL作用下的磷酸化通路分析,发现在此过程中,ERK1/2激酶的磷酸化水平明显增加,当用抑制剂U1026处理后,LDL不能促进Spl的磷酸化,也不能提高SR-BI的表达水平,说明了LDL是通过ERK1/2磷酸化通路影响Spl的磷酸化水平来激活SR-BI的。为了研究Spl的磷酸化与Spl蛋白质复合物改变的关系,我们构建了真核表达重组质粒pFlag-Sp1,并通过生物质谱的方法分析了Spl的修饰位点,结果鉴定到了Spl蛋白的多个磷酸化位点,包括一个新的Spl磷酸化位点Ser702,位于文献报道的与Spl和HDAC1结合相关的区域,并通过构建702位丝氨酸突变Flag-Sp1蛋白的方法,观察到了该位点突变后,Spl与HDAC1的结合将不受LDL的影响,证明了Spl蛋白702位丝氨酸磷酸化在SR-BI激活过程的关键作用。第二部分利用ApoE-/-动脉粥样硬化模型小鼠,建立了一套应用gel-LC-MS/MS无标记定量方法研究小鼠肝组织的差异蛋白质组的方法。结果表明,高脂饮食促使ApoE-/-小鼠的总胆固醇(Total cholesterol, TC)和LDL-C等血脂水平明显提高,主动脉粥样硬化斑块面积显著增加,说明动脉粥样硬化动物模型造模成功。正常和高脂饮食小鼠肝组织两组质谱数据分析结果共鉴定到了6677个蛋白和571个差异蛋白质,GO分类的结果中,这些差异蛋白主要参与代谢、转运等生物学过程,其中合成代谢蛋白质表达增加而分解代谢蛋白质表达降低。KEGG分析的结果表明,高脂饮食条件下,小鼠肝组织中PI3K-Akt磷酸化通路相关蛋白表达增加。建立的这套无标记定量的方法能快速简便分析动脉粥样硬化模型小鼠的蛋白质组表达水平,可用于后期动脉粥样硬化病理分子机制和候选药物的作用靶点及机制等研究。
[Abstract]:Scavenger group B receptor type I (Scavenger receptor class B type 1, SR-BI) is one of the key process of reverse cholesterol transport proteins, through selective uptake of HDL cholesterol in plasma (HDL-C) to the liver cells, regulating the level of HDL-C in the body. Therefore, regulating the expression of SR-BI in the regulation of lipid metabolism, plays an important role in reducing risk cardiovascular disease. In the first part of this study found that the high-fat diet ApoE-/- mice in liver tissue, and liver cells of HepG2 low density lipoprotein LDL treatment, the expression of SR-BI was significantly increased. In order to study the mechanism of regulation of SR-BI expression, we used the SR-BI gene promoter luciferase reporter gene and RNAi, found the transcription factor Spl plays a key role in the regulation of the process, when using the Sp1-siRNA inhibition of Spl expression, the expression of LDL can activate SR-BI. But in HepG2 cells. The expression of Spl gene had no effect on SR-BI was activated, the expression level of SR-BI decreased significantly while inhibiting the activity of Spl. The results show that a series of immune co precipitation, LDL activated SR-BI expression, changes of protein complexes of SR-BI promoter region Spl and histone deacetylase P300 and histone deacetylase HDAC1 the Sp1 raised more P300 and HDA and C1 separation, resulting in increased histone acetylation, thereby activating gene transcription. After further study of Spl protein complex changes, found in the presence of LDL, Spl protein post-translational modification changed the phosphorylation level increased significantly. By analyzing the HepG2 phosphorylation pathway in the cells under the action of LDL, it was found that the phosphorylation of ERK1/2 kinase increased significantly when treated with U1026 inhibitors, LDL can promote Spl The phosphorylation can increase the expression level of SR-BI shows that LDL is phosphorylated by ERK1/2 pathway affects the phosphorylation of Spl to activate the SR-BI. To study the relationship between Spl phosphorylation and Spl protein complexes change, we constructed a recombinant eukaryotic expression plasmid pFlag-Sp1, and analyzed the modification sites of Spl through the method of biological mass spectrometry, identification results to multiple sites of phosphorylation of Spl proteins, including a new Spl phosphorylation site of Ser702, is reported in the literature according to the relevant area with Spl and HDAC1, and through the method of constructing 702 serine mutant Flag-Sp1 protein, observed the mutation effect of combination of Spl and HDAC1, will not be affected by LDL, demonstrated that the Spl protein serine 702 phosphorylation of key activation process in SR-BI. In the second part, using the ApoE-/- model of atherosclerosis mice, established a Using the method of differential proteome of liver tissue of mice gel-LC-MS/MS label free quantitative method. The results show that the total cholesterol and high fat diet to ApoE-/- mice (Total cholesterol, TC) LDL-C and blood lipids levels increased obviously, the area of atherosclerotic plaque was significantly increased, indicating Atherosclerosis Animal Model and normal mice hyperlipidemia successfully. Diet liver tissue of two groups were identified by mass spectrometry data analysis results of protein 6677 protein and 571 different GO classification results, these proteins were mainly involved in metabolism, transport and other biological processes, including metabolism of protein synthesis expression increased protein catabolism decreased expression of the.KEGG analysis results show that the condition of high fat diet next, increase the expression of phosphorylation of PI3K-Akt pathway related protein in liver tissue of mice. The establishment of this label free quantitative method is quick and easy The proteome expression level of atherosclerotic mice is analyzed, which can be used for the study of pathological mechanism and target and mechanism of candidate drugs in later stage of atherosclerosis.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943

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