本文關鍵詞：轉(zhuǎn)錄因子Sp1對膽固醇逆轉(zhuǎn)運關鍵基因調(diào)控機制的研究　出處：《北京協(xié)和醫(yī)學院》2014年博士論文　論文類型：學位論文

  更多相關文章： 膽固醇逆轉(zhuǎn)運 SR-BI Sp1 磷酸化 轉(zhuǎn)錄調(diào)控

【摘要】：B族I型清道夫受體(Scavenger receptor class B type Ⅰ, SR-BI)是膽固醇逆轉(zhuǎn)運過程中關鍵蛋白之一,它通過選擇性攝取血漿中的HDL膽固醇(HDL-C)至肝細胞中,調(diào)節(jié)體內(nèi)HDL-C水平。因而SR-BI的表達調(diào)控對于調(diào)節(jié)血脂水平,降低心血管疾病的風險具有重要意義。本研究在第一部分發(fā)現(xiàn)了高脂飲食的ApoE-/-小鼠肝組織中,以及低密度脂蛋白LDL處理的肝細胞HepG2中,SR-BI表達均明顯增加。為了研究SR-BI表達調(diào)控的機制,我們首先采用SR-BI基因啟動子熒光素酶報告基因和RNAi等方法,發(fā)現(xiàn)了轉(zhuǎn)錄因子Spl在這一調(diào)控過程中起到關鍵作用,當用Sp1-siRNA抑制細胞Spl表達后,LDL便不能激活SR-BI的表達。但在HepG2細胞中過表達Spl對SR-BI基因并無明顯激活作用,而抑制Spl的活性后SR-BI的表達水平明顯降低。一系列的免疫共沉淀實驗表明,LDL激活SR-BI表達時,SR-BI啟動子區(qū)域Spl與組蛋白乙�；竝300和組蛋白去乙�；窰DAC1構成的蛋白質(zhì)復合物發(fā)生了改變,Sp1募集了更多的p300且同時與HDA C1分離,導致組蛋白乙酰化水平增加,從而激活基因轉(zhuǎn)錄。之后進一步考察了Spl蛋白質(zhì)復合物發(fā)生改變的原因,發(fā)現(xiàn)在LDL的作用下,Spl的蛋白質(zhì)翻譯后修飾發(fā)生了變化,其磷酸化水平明顯增加。通過對HepG2細胞在LDL作用下的磷酸化通路分析,發(fā)現(xiàn)在此過程中,ERK1/2激酶的磷酸化水平明顯增加,當用抑制劑U1026處理后,LDL不能促進Spl的磷酸化,也不能提高SR-BI的表達水平,說明了LDL是通過ERK1/2磷酸化通路影響Spl的磷酸化水平來激活SR-BI的。為了研究Spl的磷酸化與Spl蛋白質(zhì)復合物改變的關系,我們構建了真核表達重組質(zhì)粒pFlag-Sp1,并通過生物質(zhì)譜的方法分析了Spl的修飾位點,結果鑒定到了Spl蛋白的多個磷酸化位點,包括一個新的Spl磷酸化位點Ser702,位于文獻報道的與Spl和HDAC1結合相關的區(qū)域,并通過構建702位絲氨酸突變Flag-Sp1蛋白的方法,觀察到了該位點突變后,Spl與HDAC1的結合將不受LDL的影響,證明了Spl蛋白702位絲氨酸磷酸化在SR-BI激活過程的關鍵作用。第二部分利用ApoE-/-動脈粥樣硬化模型小鼠,建立了一套應用gel-LC-MS/MS無標記定量方法研究小鼠肝組織的差異蛋白質(zhì)組的方法。結果表明,高脂飲食促使ApoE-/-小鼠的總膽固醇(Total cholesterol, TC)和LDL-C等血脂水平明顯提高,主動脈粥樣硬化斑塊面積顯著增加,說明動脈粥樣硬化動物模型造模成功。正常和高脂飲食小鼠肝組織兩組質(zhì)譜數(shù)據(jù)分析結果共鑒定到了6677個蛋白和571個差異蛋白質(zhì),GO分類的結果中,這些差異蛋白主要參與代謝、轉(zhuǎn)運等生物學過程,其中合成代謝蛋白質(zhì)表達增加而分解代謝蛋白質(zhì)表達降低。KEGG分析的結果表明,高脂飲食條件下,小鼠肝組織中PI3K-Akt磷酸化通路相關蛋白表達增加。建立的這套無標記定量的方法能快速簡便分析動脈粥樣硬化模型小鼠的蛋白質(zhì)組表達水平,可用于后期動脈粥樣硬化病理分子機制和候選藥物的作用靶點及機制等研究。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R943

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