本文選題：低氧誘導(dǎo)因子-2α + 脂肪分化相關(guān)蛋白　； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景與目的 隨著人們生活水平的提高，非酒精性脂肪性肝病（non-alcoholicfatty liver disease, NAFLD）發(fā)病率逐漸升高，已經(jīng)嚴(yán)重影響人們的生活質(zhì)量。肥胖、高脂血癥、胰島素抵抗/2型糖尿病等均為NAFLD明確的易患因素。然而，近年來有文獻(xiàn)報(bào)道，阻塞性睡眠呼吸暫停綜合征(obstructive sleep apnea syndrome，OSAS)患者常伴有明顯肝損傷，認(rèn)為與呼吸暫停所致低氧誘導(dǎo)的肝臟脂肪變性有關(guān)[1-2]。目前，有關(guān)低氧情況下肝臟脂代謝異常的機(jī)制仍不十分清楚，報(bào)道甚少。本研究擬通過低氧培養(yǎng)人正常肝細(xì)胞株L02細(xì)胞，觀察肝細(xì)胞脂代謝相關(guān)固醇調(diào)節(jié)元件結(jié)合蛋白(sterol regulatory element binding protein，SREBP)-1、脂肪酸合成酶(fatty acid synthase,FAS)及低氧相關(guān)低氧誘導(dǎo)因子(hypoxia-inducible factor,HIF)2α、脂肪分化相關(guān)蛋白（adiposedifferentiation-related protein,ADFP）的表達(dá)變化，探討低氧對(duì)肝細(xì)胞脂代謝的影響及其可能機(jī)制，可能為NAFLD提供新的治療靶點(diǎn)。 方法 1、細(xì)胞培養(yǎng)：用RPMI1640培養(yǎng)液（含10％胎牛血清、0.0625g/L青霉素、0.1g/L鏈霉素）培養(yǎng)肝L02細(xì)胞。 2、實(shí)驗(yàn)分組：結(jié)合文獻(xiàn)[3]，實(shí)驗(yàn)分對(duì)照組（21%O_2常氧培養(yǎng)）、低氧組（1%O_2低氧培養(yǎng)）。 3、低氧模型的建立：MTT比色法檢測1%低氧對(duì)L02細(xì)胞生長的影響，確定低氧培養(yǎng)時(shí)間點(diǎn)。 4、通過L02細(xì)胞內(nèi)TG測定和油紅O染色了解其脂變情況； RT-PCR法檢測HIF-2α、SREBP-1mRNA的表達(dá)；Western Blot法檢測SREBP-1、FAS、HIF-2α、ADFP的蛋白表達(dá)。 結(jié)果 1、成功建立低氧誘導(dǎo)的肝L02細(xì)胞脂肪變性模型。隨著低氧暴露時(shí)間的不斷延長，肝細(xì)胞脂肪變程度逐漸加重。 1）低氧模型的建立：將細(xì)胞放入溫度為37℃含有1%O_2、5％CO_2、94%N2飽和濕度的三氣培養(yǎng)箱中分別培養(yǎng)12、24、48h。 2）TG測定：與對(duì)照組相應(yīng)時(shí)間點(diǎn)比較，低氧處理各時(shí)間點(diǎn)TG含量均增加，其中12h點(diǎn)，兩組差異無統(tǒng)計(jì)學(xué)意義（P0.05），，24、48h兩個(gè)時(shí)間點(diǎn)，差異有統(tǒng)計(jì)學(xué)意義（P0.01）；且低氧處理時(shí)間越長，TG含量越多（F=115.04，P0.01）。 3）油紅O染色：對(duì)照組僅見胞質(zhì)內(nèi)少量橘紅色脂滴，而各低氧處理組隨著低氧培養(yǎng)時(shí)間延長，細(xì)胞內(nèi)脂滴數(shù)量增多，且出現(xiàn)脂滴融合現(xiàn)象。 2、各組細(xì)胞內(nèi)SREBP-1、FAS的表達(dá)情況。 低氧12、24、48h各時(shí)間點(diǎn)SREBP-1mRNA和蛋白、FAS蛋白表達(dá)均較對(duì)照組下調(diào)（P值均0.01）。 3、各組細(xì)胞內(nèi)HIF-2α、ADFP的表達(dá)變化。 各時(shí)間點(diǎn)細(xì)胞內(nèi)均可檢測到HIF-2αmRNA的表達(dá)，但差異無統(tǒng)計(jì)學(xué)意義。常氧組細(xì)胞中未檢測到HIF-2α蛋白表達(dá)，低氧處理3h后HIF-2α蛋白表達(dá)逐漸增加，6h后達(dá)高峰，12、24h又逐漸降低（F=85.30,P0.01）；與對(duì)照組比較，低氧12、24、48h各時(shí)間點(diǎn)ADFP蛋白表達(dá)均明顯上調(diào)（P值均0.05），且隨低氧時(shí)間延長，表達(dá)量逐漸增多（F=167.49，P0.05）。 結(jié)論 1、SREBP-1-FAS生脂途徑可能沒有參與低氧介導(dǎo)的肝細(xì)胞脂變。 2、HIF-2-ADFP是介導(dǎo)低氧誘導(dǎo)肝細(xì)胞脂質(zhì)沉積的重要機(jī)制。 3、HIF-2α有可能成為脂肪肝治療的新靶點(diǎn)。
[Abstract]:Background and purpose of study

In recent years , it has been reported that the mechanism of lipid metabolism related sterol regulatory element binding protein , fatty acid synthase ( FAS ) and hypoxia - related hypoxia - inducible factor ( ADFP ) in patients with obstructive sleep apnea syndrome ( Obstructive sleep apnea syndrome ) have been reported in recent years . The effects of hypoxia on lipid metabolism in liver cells and possible mechanism of hypoxia - related hypoxia - inducible factor ( ADFP ) may be observed .

method

1 . Cell culture : The liver L02 cells were cultured with RPMI1640 medium ( containing 10 % fetal bovine serum , 0.0625 g / L penicillin , 0.1 g / L streptomycin ) .

2 . Experimental group : The experiment group was divided into control group ( 21 % O _ 2 normal oxygen culture ) and hypoxia group ( 1 % O _ 2 hypoxia culture ) .

3 . The hypoxia model was established : MTT colorimetric assay was used to detect the effect of 1 % hypoxia on the growth of L02 cells and to determine the time point of hypoxia culture .

4 . L02 intracellular TG assay and oil red O staining were used to investigate the lipid change .
RT - PCR assay was used to detect the expression of HIF - 2偽and BIR - 1mRNA .
Western Blot was used to detect the expression of protein , FAS , HIF - 2偽and ADFP .

Results

1 . The fat degeneration model of L02 cells induced by hypoxia was successfully established . With the increasing of hypoxia exposure time , the degree of fatty degeneration of hepatocytes was gradually increased .

1 ) Establishment of hypoxic model : The cells were cultured in a three - gas incubator containing 1 % O2 , 5 % CO _ 2 , 94 % N2 saturated humidity at 37 鈩

本文編號(hào)：2036742