【摘要】：目的：非酒精性脂肪肝�。╪on-alcoholic fatty liver disease，NAFLD）已經(jīng)成為危害人類健康的主要代謝性疾病之一，但其發(fā)病機制尚不完全清楚。高脂及炎癥狀態(tài)均可以作為獨立的危險因素導(dǎo)致NAFLD的發(fā)生發(fā)展。CD36屬于B族清道夫受體，可以介導(dǎo)長鏈脂肪酸及氧化低密度脂蛋白的跨膜轉(zhuǎn)運，其表達(dá)量與肝臟脂肪變性程度密切相關(guān)。哺乳動物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）是一種高度保守的絲氨酸/蘇氨酸激酶，它在調(diào)節(jié)細(xì)胞生長、增殖及蛋白質(zhì)翻譯中起著重要的作用。本課題旨在研究高脂及炎癥狀態(tài)是否可以通過激活mTOR信號通路導(dǎo)致CD36翻譯效率升高，最終引起CD36蛋白表達(dá)量及肝細(xì)胞內(nèi)脂質(zhì)蓄積增加。 方法：使用軟脂酸、TNF-及IL-6處理人肝癌細(xì)胞株HepG2，高脂飼料喂養(yǎng)及酪蛋白皮下注射C57BL/6J小鼠的方法建立體外及體內(nèi)高脂及炎癥模型。HepG2細(xì)胞分為：對照組1（0.2%BSA）、軟脂酸處理組（0.2%BSA+0.08mmol/L軟脂酸）、對照組2（0.2%BSA+0.04mmol/L軟脂酸）、TNF-處理組（0.2%BSA+0.04mmol/L軟脂酸+25ng/mLTNF-）、IL-6處理組（0.2%BSA+0.04mmol/L軟脂酸+20ng/mLIL-6），處理時間為24小時。雄性C57BL/6J小鼠分為：正常飼料組（正常飼料）、高脂飼料組（高脂飼料）、正常飼料+酪蛋白處理組（正常飼料+0.5mL10%酪蛋白皮下注射），，建模時間為14周。油紅O染色觀察肝細(xì)胞內(nèi)脂質(zhì)蓄積的情況。酶聯(lián)免疫吸附試驗及酶偶聯(lián)比色法分別檢測肝細(xì)胞內(nèi)游離脂肪酸（FFA）及甘油三酯（TG）含量。Real-TimePCR檢測肝細(xì)胞CD36mRNA表達(dá)。Western Blotting檢測肝細(xì)胞CD36蛋白表達(dá)，mTOR及其下游翻譯調(diào)控蛋白：核糖體蛋白p70S6激酶（p70ribosomal protein S6kinase，p70S6K）、eIF4E結(jié)合蛋白1（eukaryoticinitiation factor4E-binding protein1，4E-BP1）及真核翻譯起始因子4E（eukaryotic initiation factor4E，eIF4E）的磷酸化水平。蛋白降解實驗檢測肝細(xì)胞CD36蛋白穩(wěn)定性。多聚核糖體分析檢測肝細(xì)胞CD36翻譯效率。并應(yīng)用mTOR特異性抑制劑雷帕霉素作用于高脂及炎癥狀態(tài)下的HepG2細(xì)胞（10ng/mL雷帕霉素）及C57BL/6J小鼠（2mg/kg體重的雷帕霉素皮下注射），觀察其對上述檢測指標(biāo)的影響。 結(jié)果：油紅O染色、FFA及TG定量檢測發(fā)現(xiàn)高脂及炎癥狀態(tài)能使HepG2細(xì)胞及C57BL/6J小鼠肝臟組織脂質(zhì)蓄積增加（P0.05）。Western Blotting及Real-Time PCR檢測發(fā)現(xiàn)高脂及炎癥狀態(tài)能使CD36蛋白表達(dá)量升高（P0.05），而CD36蛋白表達(dá)量卻沒有明顯變化（P0.05）。蛋白降解實驗發(fā)現(xiàn)高脂及炎癥狀態(tài)并沒有改變HepG2細(xì)胞CD36蛋白穩(wěn)定性（P0.05）。多聚核糖體分析發(fā)現(xiàn)高脂及炎癥狀態(tài)可以使CD36翻譯效率明顯升高。進(jìn)一步行Western Blotting檢測發(fā)現(xiàn)高脂及炎癥狀態(tài)可以升高mTOR及其下游翻譯調(diào)控蛋白p70S6K、4EBP-1及eIF4E的磷酸化水平（P0.05）。加入雷帕霉素之后，相應(yīng)的高脂及炎癥處理組mTOR信號通路磷酸化水平降低（P0.05），導(dǎo)致CD36翻譯效率及蛋白表達(dá)量減少（P0.05），最終引起HepG2細(xì)胞及C57BL/6J小鼠肝臟組織脂質(zhì)蓄積減輕（P0.05）。 結(jié)論：高脂及炎癥狀態(tài)可以激活HepG2細(xì)胞及C57BL/6J小鼠肝臟組織mTOR信號通路導(dǎo)致肝臟CD36翻譯效率升高，最終引起CD36蛋白表達(dá)及脂質(zhì)蓄積增加。
[Abstract]:Objective: Non-alcoholic fatty liver disease (NAFLD) has become one of the major metabolic diseases endangering human health, but its pathogenesis is not fully understood. High fat and inflammatory state can be used as independent risk factors leading to the occurrence and development of NAFLD. CD36 belongs to B scavenger receptor and can mediate the development of NAFLD. Long-chain fatty acids and oxidized low-density lipoproteins are transmembrane transporters whose expression is closely related to the degree of liver steatosis. Mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase, which plays an important role in regulating cell growth, proliferation and protein translation. The purpose of this study was to investigate whether hyperlipidemia and inflammation could induce the increase of CD36 translation efficiency by activating the mTOR signaling pathway, and ultimately lead to the increase of CD36 protein expression and lipid accumulation in hepatocytes.
METHODS: Human hepatoma cell line HepG2 was treated with palmitic acid, TNF-and IL-6, and high-fat diet and subcutaneous injection of casein into C57BL/6J mice. HepG2 cells were divided into control group 1 (0.2% BSA), palmitic acid treatment group (0.2% BSA + 0.08mmol/L palmitic acid), control group 2 (0.2% BSA + 0.04 mmol/L palmitic acid). Acid, TNF-treated group (0.2% BSA + 0.04 mmol/L palmitic acid + 25 ng/mLTNF-), IL-6 treated group (0.2% BSA + 0.04 mmol/L palmitic acid + 20 ng/mLIL-6), treatment time was 24 hours. Male C57BL/6J mice were divided into normal diet group (normal diet), high-fat diet group (high-fat diet), normal diet + casein treated group (normal diet + 0.5 mL 10% casein subcutaneous injection). The content of free fatty acid (FFA) and triglyceride (TG) in hepatocytes were detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-coupled colorimetry (ELISA). The expression of CD36 mRNA in hepatocytes was detected by Real-Time PCR. The expression of CD36 protein in hepatocytes was detected by Western Blotting. The expression of CD36 protein in hepatocytes was detected by mTOR and its subunits. The phosphorylation levels of p70S6 kinase (p70ribosomal protein S6kinase, p70S6K), eIF4E binding protein 1, 4E-BP1 and eukaryotic translation initiation factor 4E (eIF4E) in hepatocytes were determined by protein degradation assay. Sex. Polyribosome analysis was used to detect the translation efficiency of CD36 in hepatocytes. Rapamycin, a specific inhibitor of mTOR, was used to treat high-fat and inflammatory HepG2 cells (10ng/mL rapamycin) and C57BL/6J mice (2mg/kg body weight rapamycin subcutaneously) to observe the effects of rapamycin on the above indexes.
Results: Oil red O staining, FFA and TG quantitative detection showed that high fat and inflammation could increase the lipid accumulation of HepG2 cells and C57BL/6J mice liver tissue (P 0.05). Western Blotting and Real-Time PCR detection showed that high fat and inflammation could increase the expression of CD36 protein (P 0.05), but the expression of CD36 protein did not change significantly (P 0.05). Protein degradation assay showed that high fat and inflammation did not change the stability of CD36 protein in HepG2 cells (P 0.05). Polyribosome analysis showed that high fat and inflammation could significantly increase the translation efficiency of CD36. Phosphorylation levels of EBP-1 and eIF4E (P 0.05). When rapamycin was added, the phosphorylation levels of mTOR signaling pathway decreased (P 0.05) in the corresponding hyperlipidemia and inflammation treatment groups, resulting in the decrease of CD36 translation efficiency and protein expression (P 0.05), and ultimately the decrease of lipid accumulation in HepG2 cells and C57BL/6J mice liver tissue (P 0.05).
CONCLUSION: Hyperlipidemia and inflammation can activate the mTOR signaling pathway in HepG2 cells and C57BL/6J mice liver tissues, resulting in an increase in the efficiency of liver CD36 translation, and ultimately in the expression of CD36 protein and lipid accumulation.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575.5

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