【摘要】：胞漿硫酸基轉(zhuǎn)移酶(SULT) 2B1b催化25-羥化膽固醇(25HC)硫酸化,反應(yīng)主要產(chǎn)物為3-硫酸-25-羥化膽固醇(25HC3S)。研究發(fā)現(xiàn)該硫酸化產(chǎn)物可通過(guò)抑制LXR/SREBPs信號(hào)轉(zhuǎn)導(dǎo)通路降低THP-1來(lái)源的巨噬細(xì)胞內(nèi)脂質(zhì)水平。因此,合成25HC3S的關(guān)鍵限速酶SULT2B1b可能在調(diào)節(jié)脂質(zhì)代謝中起重要作用。本實(shí)驗(yàn)中,我們?cè)谠酥鲃?dòng)脈內(nèi)皮細(xì)胞內(nèi)研究SULT2B1b對(duì)脂質(zhì)代謝的作用及機(jī)制,并在小鼠非酒精性脂肪性肝病模型中檢測(cè)SULT2B1b的表達(dá)及其重要的生理意義。 本研究共包括三部分內(nèi)容,詳細(xì)如下： 第一部分：預(yù)實(shí)驗(yàn)及其它文獻(xiàn)報(bào)道,羥基類固醇硫酸基轉(zhuǎn)移酶(SULT2B1b)同樣表達(dá)于原代人主動(dòng)脈內(nèi)皮細(xì)胞及小鼠肝臟組織內(nèi),但表達(dá)量較低。為了更好的研究SULT2B1b及其所產(chǎn)生的內(nèi)源性硫化固醇對(duì)脂質(zhì)代謝的作用及機(jī)制,在這些細(xì)胞或組織內(nèi)過(guò)表達(dá)SULT2B1b是一種比較合理的方法,所以我們構(gòu)建含SULT2B1b基因的腺病毒載體。 第二部分：利用我們所構(gòu)建的含SULT2B1b基因的腺病毒載體轉(zhuǎn)染原代人主動(dòng)脈內(nèi)皮細(xì)胞,研究SULT2B1b對(duì)脂質(zhì)代謝的影響。選用該細(xì)胞系作為我們的研究對(duì)象,是因?yàn)檠軆?nèi)皮細(xì)胞在動(dòng)脈粥樣硬化等脂質(zhì)代謝紊亂相關(guān)疾病中發(fā)揮重要作用。結(jié)果顯示,腺病毒(Ad-SULT2B1b)感染后,SULT2B1b基因在原代人主動(dòng)脈內(nèi)皮細(xì)胞內(nèi)成功過(guò)表達(dá)；加入[H3]標(biāo)記的25-羥化膽固醇(25HC)追蹤其代謝變化,2小時(shí)后約40%25HC被硫酸化,24小時(shí)后大于50%25HC被硫酸化,HPLC鑒定硫酸化產(chǎn)物發(fā)現(xiàn)主要為3-硫酸-25-羥化膽固醇(25HC3S)；進(jìn)一步檢測(cè)細(xì)胞內(nèi)脂質(zhì)水平的變化,發(fā)現(xiàn)在25HC存在的情況下,過(guò)表達(dá)SULT2B1b顯著降低細(xì)胞內(nèi)甘油三酯、總膽固醇、游離膽固醇及游離脂肪酸,并降低脂質(zhì)合成相關(guān)基因,包括LXRα、SREBP-1、ACC1、FAS和HMGR等蛋白及mRNA水平；然而在沒有25-羥化膽固醇存在,或在T0901317 (化學(xué)合成的LXR激動(dòng)劑)存在的情況下,過(guò)表達(dá)SULT2B1b降脂作用不明顯。表明SULT2B1b的降脂作用主要是通過(guò)其形成的內(nèi)源性25HC3S而產(chǎn)生的,并非其自身直接作用。另外,我們還在相對(duì)高表達(dá)SULT2B1b的HepG2細(xì)胞內(nèi),采用干擾RNA技術(shù)敲除SULT2B1b基因的表達(dá),反面驗(yàn)證氧化固醇硫酸化的降脂作用。 第三部分：高脂或高膽固醇飼養(yǎng)C57BL/6及LDLR-/-敲基因小鼠建立非酒精性脂肪性肝病模型,通過(guò)尾靜脈注射含SULT2B1b基因的腺病毒,研究其在小鼠體內(nèi)對(duì)脂質(zhì)代謝的作用。小鼠被隨機(jī)分為三組,即C57BL/6小鼠高膽固醇飼養(yǎng)組,C57BL/6小鼠高膽固醇飼養(yǎng)并腹腔注射25HC組,LDLR-/-敲基因小鼠高脂飼養(yǎng)組。每組又分為感染SULT2B1b和對(duì)照病毒β-Gal組。每小組5-6只小鼠。結(jié)果顯示,過(guò)表達(dá)SULT2B1b的小鼠血清及肝臟組織內(nèi)脂質(zhì)水平(包括甘油三酯、膽固醇及游離脂肪酸)與相應(yīng)組別內(nèi)的對(duì)照病毒組明顯下降,其中C57BL/6小鼠高膽固醇飼養(yǎng)并腹腔注射25HC組及LDLR-/-敲基因小鼠高脂飼養(yǎng)組更為明顯。HPLC分析肝臟內(nèi)氧化固醇及硫酸化的氧化固醇發(fā)現(xiàn),C57BL/6小鼠高膽固醇飼養(yǎng)并腹腔注射25HC組過(guò)表達(dá)SULT2B1b后,氧化固醇明顯下降而硫酸化的氧化固醇則明顯升高,而沒有腹腔注射25HC的小鼠變化不明顯。脂質(zhì)相關(guān)基因檢測(cè)發(fā)現(xiàn)過(guò)表達(dá)SULT2B1b后,LXRα、SREBP-1、ACC1和FAS蛋白及mRNA水平均明顯下降。另外,我們也分離了小鼠主動(dòng)脈,正常C57BL/6小鼠飼養(yǎng)高膽固醇10周后均未檢測(cè)到動(dòng)脈粥樣硬化,而在LDLR-/-敲基因小鼠內(nèi)轉(zhuǎn)染對(duì)照病毒組可見脂質(zhì)堆積和動(dòng)脈粥樣硬化,過(guò)表達(dá)SULT2Blb后,動(dòng)脈粥樣硬化現(xiàn)象消失。 綜上所述,羥基類固醇硫酸基轉(zhuǎn)移酶(SULT2Blb)通過(guò)硫酸化氧化固醇抑制LXR/SREBPs脂質(zhì)合成途徑,從而有效降低細(xì)胞內(nèi)、小鼠血清及肝臟組織內(nèi)脂質(zhì)水平,在脂質(zhì)代謝中發(fā)揮重要作用。這些發(fā)現(xiàn)也為脂質(zhì)代謝紊亂相關(guān)疾病如動(dòng)脈粥樣硬化及非酒精性脂肪性肝病等的防治提供了新的理論依據(jù)及途徑。
[Abstract]:Cytoplasmic sulfate transferase (SULT) 2B1b catalyzes the sulfation of 25-hydroxycholesterol (25HC) with the main product of 3-sulfuric acid-25-hydroxycholesterol (25HC3S). It has been found that the sulfated product can reduce lipid levels in THP-1-derived macrophages by inhibiting LXR/SREBPs signal transduction pathway. Therefore, the key rate-limiting enzyme for the synthesis of 25HC3S, SULT2B1b In this study, we studied the effect and mechanism of SULT2B1b on lipid metabolism in primary human aortic endothelial cells, and detected the expression of SULT2B1b in non-alcoholic fatty liver disease model in mice.
This study consists of three parts, as follows:
Part I: Preliminary studies and other literature have reported that hydroxysteroid sulfate transferase (SULT2B1b) is also expressed in primary human aortic endothelial cells and mouse liver tissues, but the expression is low. Overexpression of SULT2B1b in tissues is a reasonable method, so we constructed adenovirus vector containing SULT2B1b gene.
The second part is to study the effect of SULT2B1b on lipid metabolism by transfecting primary human aortic endothelial cells with adenovirus vector containing SULT2B1b gene. The results showed that SULT2B1b gene was overexpressed in primary human aortic endothelial cells after adenovirus (Ad-SULT2B1b) infection, and 25-hydroxycholesterol (25HC) labeled with [H3] was added to track its metabolic changes. About 40% of 25HC was sulfated 2 hours later, and more than 50% of 25HC was sulfated 24 hours later. - Hydroxycholesterol (25HC3S) was further detected, and the expression of SULT2B1b significantly decreased triglycerides, total cholesterol, free cholesterol and free fatty acids in the cells in the presence of 25HC, and reduced the levels of protein and mRNA related to lipid synthesis, including LXR alpha, SREBP-1, ACC1, FAS and HMGR. However, in the absence of 25-hydroxycholesterol, or in the presence of T0901317 (a chemically synthesized LXR agonist), the lipid-lowering effect of SULT2B1b is not obvious. This suggests that the lipid-lowering effect of SULT2B1b is mainly produced by the endogenous 25HC3S formed by SULT2B1b, but not directly by itself. In HepG2 cells, the expression of SULT2B1b gene was knocked out by interfering RNA technology, and the lipid-lowering effect of oxidized sterol sulfonation was tested.
Part III: Non-alcoholic fatty liver disease models were established in C57BL/6 and LDLR-/-knocking mice fed with high-fat or high-cholesterol diet. Adenoviruses containing SULT2B1b gene were injected into tail vein to study their effects on lipid metabolism in mice. Mice were randomly divided into three groups: C57BL/6 mice fed with high-cholesterol diet and C57BL/6 mice fed with high-cholesterol diet. The serum and liver lipid levels (including triglycerides, cholesterol and free fatty acids) in mice overexpressing SULT2B1b were compared with those in the corresponding groups. The levels of serum oxysterol in C57BL/6 mice were significantly lower than those in C57BL/6 mice fed with high cholesterol and intraperitoneally injected with 25HC and LDLR-/-knocking mice fed with high cholesterol. The levels of LXR-alpha, SREBP-1, ACC 1 and FAS protein and mRNA were significantly decreased after the expression of SULT2B1b. In addition, the aorta of normal C57BL/6 mice were isolated and the cholesterol levels of normal C57BL/6 mice were not detected after 10 weeks. Atherosclerosis was detected, and lipid accumulation and atherosclerosis were observed in the LDLR-/-knocking mice transfected with the control virus. After overexpression of SULT2Blb, atherosclerosis disappeared.
To sum up, hydroxysteroid sulfate transferase (SULT2Blb) inhibits LXR/SREBPs lipid biosynthesis via sulfated oxidative steroids, thereby effectively reducing lipid levels in cells, mouse serum and liver tissues, and plays an important role in lipid metabolism. These findings also play an important role in lipid metabolism disorders related diseases such as atherosclerosis and atherosclerosis. The prevention and treatment of nonalcoholic fatty liver disease provide a new theoretical basis and approach.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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