本文關(guān)鍵詞：轉(zhuǎn)錄因子Sp1對(duì)膽固醇逆轉(zhuǎn)運(yùn)關(guān)鍵基因調(diào)控機(jī)制的研究　出處：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 膽固醇逆轉(zhuǎn)運(yùn) 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(yùn)過程中關(guān)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(xì)胞中,調(diào)節(jié)體內(nèi)HDL-C水平。因而SR-BI的表達(dá)調(diào)控對(duì)于調(diào)節(jié)血脂水平,降低心血管疾病的風(fēng)險(xiǎn)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(xì)胞HepG2中,SR-BI表達(dá)均明顯增加。為了研究SR-BI表達(dá)調(diào)控的機(jī)制,我們首先采用SR-BI基因啟動(dòng)子熒光素酶報(bào)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(guān)鍵作用,當(dāng)用Sp1-siRNA抑制細(xì)胞Spl表達(dá)后,LDL便不能激活SR-BI的表達(dá)。但在HepG2細(xì)胞中過表達(dá)Spl對(duì)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(dá)水平明顯降低。一系列的免疫共沉淀實(shí)驗(yàn)表明,LDL激活SR-BI表達(dá)時(shí),SR-BI啟動(dòng)子區(qū)域Spl與組蛋白乙�；竝300和組蛋白去乙�；窰DAC1構(gòu)成的蛋白質(zhì)復(fù)合物發(fā)生了改變,Sp1募集了更多的p300且同時(shí)與HDA C1分離,導(dǎo)致組蛋白乙酰化水平增加,從而激活基因轉(zhuǎn)錄。之后進(jìn)一步考察了Spl蛋白質(zhì)復(fù)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(duì)HepG2細(xì)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(dāng)用抑制劑U1026處理后,LDL不能促進(jìn)Spl的磷酸化,也不能提高SR-BI的表達(dá)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(fù)合物改變的關(guān)系,我們構(gòu)建了真核表達(dá)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(diǎn),結(jié)果鑒定到了Spl蛋白的多個(gè)磷酸化位點(diǎn),包括一個(gè)新的Spl磷酸化位點(diǎn)Ser702,位于文獻(xiàn)報(bào)道的與Spl和HDAC1結(jié)合相關(guān)的區(qū)域,并通過構(gòu)建702位絲氨酸突變Flag-Sp1蛋白的方法,觀察到了該位點(diǎn)突變后,Spl與HDAC1的結(jié)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(guān)鍵作用。第二部分利用ApoE-/-動(dòng)脈粥樣硬化模型小鼠,建立了一套應(yīng)用gel-LC-MS/MS無標(biāo)記定量方法研究小鼠肝組織的差異蛋白質(zhì)組的方法。結(jié)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(dòng)脈粥樣硬化斑塊面積顯著增加,說明動(dòng)脈粥樣硬化動(dòng)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(jié)果共鑒定到了6677個(gè)蛋白和571個(gè)差異蛋白質(zhì),GO分類的結(jié)果中,這些差異蛋白主要參與代謝、轉(zhuǎn)運(yùn)等生物學(xué)過程,其中合成代謝蛋白質(zhì)表達(dá)增加而分解代謝蛋白質(zhì)表達(dá)降低。KEGG分析的結(jié)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(guān)蛋白表達(dá)增加。建立的這套無標(biāo)記定量的方法能快速簡(jiǎn)便分析動(dòng)脈粥樣硬化模型小鼠的蛋白質(zhì)組表達(dá)水平,可用于后期動(dòng)脈粥樣硬化病理分子機(jī)制和候選藥物的作用靶點(diǎn)及機(jī)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(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

【相似文獻(xiàn)】

相關(guān)會(huì)議論文 前3條

1 董靜;趙水平;;煙酸促進(jìn)膽固醇逆轉(zhuǎn)運(yùn)在體和離體研究[A];中華醫(yī)學(xué)會(huì)心血管病學(xué)分會(huì)第十次全國(guó)心血管病學(xué)術(shù)會(huì)議匯編[C];2008年

2 廖端芳;;Caveolin-1對(duì)細(xì)胞膽固醇逆轉(zhuǎn)運(yùn)的調(diào)節(jié)作用[A];湖南省生理科學(xué)會(huì)新世紀(jì)學(xué)術(shù)年會(huì)論文摘要匯編[C];2002年

3 張弛;尹衛(wèi)東;肖俊霞;李勤凱;蔡曼波;劉毅;侯洪杰;;NO-1886調(diào)節(jié)膽固醇逆轉(zhuǎn)運(yùn)相關(guān)基因抑制動(dòng)脈粥樣硬化的作用[A];第八次全國(guó)動(dòng)脈硬化性疾病學(xué)術(shù)會(huì)議論文集[C];2005年

相關(guān)博士學(xué)位論文 前4條

1 楊帆;轉(zhuǎn)錄因子Sp1對(duì)膽固醇逆轉(zhuǎn)運(yùn)關(guān)鍵基因調(diào)控機(jī)制的研究[D];北京協(xié)和醫(yī)學(xué)院;2014年

2 樂薇;電針豐隆對(duì)高脂血癥模型大鼠膽固醇逆轉(zhuǎn)運(yùn)通路的影響[D];湖北中醫(yī)藥大學(xué);2013年

3 高潔;以ABCA1為靶點(diǎn)的新型抗動(dòng)脈粥樣硬化藥物篩選模型的構(gòu)建與應(yīng)用研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2008年

4 董靜;煙酸促進(jìn)膽固醇逆轉(zhuǎn)運(yùn)在體和離體研究[D];中南大學(xué);2008年

相關(guān)碩士學(xué)位論文 前5條

1 張新勝;中鏈甘油三酯對(duì)膽固醇逆轉(zhuǎn)運(yùn)的影響及其機(jī)制研究[D];中國(guó)人民解放軍醫(yī)學(xué)院;2015年

2 郭瑩瑩;脂聯(lián)素對(duì)APN-/-小鼠體內(nèi)巨噬細(xì)胞膽固醇逆轉(zhuǎn)運(yùn)的影響及其作用機(jī)制[D];山西醫(yī)科大學(xué);2015年

3 高曉玲;抑郁對(duì)膽固醇逆轉(zhuǎn)運(yùn)的影響及相關(guān)的分子機(jī)制[D];廣州中醫(yī)藥大學(xué);2014年

4 張弛;NO-1886調(diào)節(jié)膽固醇逆轉(zhuǎn)運(yùn)相關(guān)基因抑制動(dòng)脈粥樣硬化的作用[D];南華大學(xué);2005年

5 王晉鋒;降脂藥物對(duì)B族Ⅰ型清道夫受體表達(dá)和功能的影響[D];中國(guó)協(xié)和醫(yī)科大學(xué);2007年

，

本文編號(hào)：1440372