本文關(guān)鍵詞： 低氧誘導(dǎo)因子-1 乙醛 肝星狀細(xì)胞 α-平滑肌肌動蛋白 酒精性肝纖維化　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:酒精性肝病(alcoholic liver disease,ALD)是因過量攝入酒精引發(fā)的中毒性肝臟病變,早期通常表現(xiàn)為酒精性脂肪肝(alcoholic fatty liver disease,AFLD)可進(jìn)展為酒精性肝炎、酒精性肝纖維化(alcohol liver fibrosis,ALF)、肝硬化、甚至肝癌。ALF病因明確,但其發(fā)病機(jī)制尚未完全清楚。目前認(rèn)為肝星狀細(xì)胞(hepatic stellate cells,HSCs)增殖與活化是肝纖維化發(fā)生的核心環(huán)節(jié)。近十多年來國內(nèi)外研究表明,缺氧誘導(dǎo)因子-1(hypoxia inducible factor-1,HIF-1)作為低氧應(yīng)答調(diào)控的管家基因,可能通過其調(diào)節(jié)的靶基因參與HSCs的活化與增殖。HIF-1是由調(diào)節(jié)亞基HIF-1α與結(jié)構(gòu)性亞基HIF-1β共同組成的異源二聚體,HIF-1α對HIF-1發(fā)揮轉(zhuǎn)錄因子活性起著至關(guān)重要的影響,在細(xì)胞內(nèi)水平受到氧依賴和非氧依賴信號事件的調(diào)控。有研究者用內(nèi)毒素培育人肝星狀細(xì)胞系,發(fā)現(xiàn)HIF-1α高表達(dá),且標(biāo)志HSCs活化的I型膠原蛋白(Collagen I)、α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)高表達(dá),抑制HIF-1α表達(dá)后,I型膠原蛋白及α-SMA表達(dá)也隨之減低。我們前期在酒精灌胃構(gòu)建的大鼠ALD模型中觀察到:隨著造模時間的延長,肝組織HIF-1αmRNA和蛋白表達(dá)隨之增多,提示HIF-1α可能參與著ALD發(fā)病過程。本研究選用200μmol/L乙醛刺激大鼠HSC-T6細(xì)胞,特異性抑制HIF-1α表達(dá),觀察HSC-T6細(xì)胞增殖情況及α-SMA和Collagen I表達(dá)變化。旨在細(xì)胞水平闡明抑制HIF-1α表達(dá)對HSC-T6細(xì)胞增殖及α-SMA和Collagen I表達(dá)影響,探討HIF-1α調(diào)控的HSCs在ALF中的作用。方法:復(fù)蘇HSC-T6細(xì)胞用含10%胎牛血清、1%青鏈霉素的高糖DMEM培養(yǎng)基在5%CO2,37℃條件下培養(yǎng)到對數(shù)生長期。實(shí)驗(yàn)分為正常對照組(常規(guī)培養(yǎng))、乙醛刺激組(終濃度為200μmol/L)、HIF-1αsiRNA組(si RNA100nm/L+乙醛終濃度為200μmol/L)、HIF-1αsiRNA陰性對照(negative control,NC)組(NC siRNA+乙醛終濃度為200μmol/L)。利用200μmol/L乙醛刺激大鼠HSC-T6細(xì)胞,將HIF-1αsi RNA、NC siRNA通過脂質(zhì)體lipofectaminetm2000瞬時轉(zhuǎn)染至hsc-t6細(xì)胞,培養(yǎng)6h后,更換為新鮮完全培養(yǎng)液繼續(xù)培養(yǎng)48h,應(yīng)用cck-8檢測大鼠hsc-t6細(xì)胞增殖變化;收集細(xì)胞,分別利用rt-qpcr和westernblot法檢測hif-1α、α-sma和collagenimrna和蛋白表達(dá)水平;用4%多聚甲醛固定,行免疫熒光染色,觀察α-sma蛋白表達(dá)情況。結(jié)果:1轉(zhuǎn)染hif-1αsirna敲低hif-1α基因?qū)Υ笫骽sc-t6細(xì)胞增殖的影響瞬時轉(zhuǎn)染hif-1αsirna48h后,細(xì)胞增長情況明顯減慢,cck-8比色結(jié)果:與正常對照組比較,乙醛刺激組od值明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.01),提示200μmol/l乙醛可明顯促進(jìn)hsc-t6細(xì)胞增殖;hif-1αsirna組od值與乙醛刺激組比較,細(xì)胞增長情況明顯減慢,且差異具有統(tǒng)計(jì)學(xué)意義(p0.01);hif-1αsirnanc組與乙醛刺激組比較,od值差異無統(tǒng)計(jì)學(xué)意義(p均0.05)。2采用rt-qpcr法檢測hif-1α、α-sma和collagenⅠ基因相對于β-action內(nèi)參基因表達(dá)變化:正常對照組hsc-t6細(xì)胞中hif-1α、α-sma和collagenⅠ的表達(dá)均較少;乙醛刺激組與正常對照組比較,hif-1α、α-sma和collagenⅠ表達(dá)量明顯增多(p均0.01);敲低hif-1α基因,hif-1αsirna組與乙醛刺激組比較,hif-1α表達(dá)降低的同時α-sma和collagenⅠ表達(dá)明顯減少(p均0.01);hif-1αsirnanc組和乙醛刺激組比較,各個指標(biāo)表達(dá)的變化,差異均無統(tǒng)計(jì)學(xué)意義(p均0.05)。3通過westernblot法檢測hif-1αsirna轉(zhuǎn)染后hsc-t6細(xì)胞中的hif-1α、α-sma和collagenⅠ蛋白水平表達(dá)變化:乙醛刺激組hif-1α、α-sma和collagenⅠ蛋白表達(dá)量明顯增多,乙醛刺激組與正常對照組比較差異有統(tǒng)計(jì)學(xué)意義(p均0.01);轉(zhuǎn)染hif-1αsirna48h后,hif-1α蛋白表達(dá)降低,與乙醛刺激組比較差異具有統(tǒng)計(jì)學(xué)意義(p0.01),且α-sma和collagenⅠ蛋白的表達(dá)量也減低;hif-1αsirnanc組與乙醛刺激組比較,各個指標(biāo)表達(dá)變化,差異均無統(tǒng)計(jì)學(xué)意義(p均0.05)。4免疫熒光染色法觀察大鼠hsc-t6細(xì)胞中α-sma蛋白表達(dá)情況:可觀察到正常對照組細(xì)胞表達(dá)少量α-sma;乙醛刺激組細(xì)胞α-sma蛋白表達(dá)增多;轉(zhuǎn)染hif-1αsirna48h后,細(xì)胞中的α-sma蛋白表達(dá)減少。結(jié)論:乙醛能夠促進(jìn)肝星狀細(xì)胞增殖活化,敲低HIF-1α對肝星狀細(xì)胞的增殖活化有抑制作用,可能是酒精性肝纖維化的潛在治療靶點(diǎn)。
[Abstract]:Objective: alcoholic liver disease (alcoholic liver, disease, ALD) is due to excessive intake of alcohol poisoning caused by liver lesions, early is often in the form of alcoholic fatty liver (alcoholic fatty liver disease, AFLD) in alcoholic hepatitis, alcoholic liver fibrosis (alcohol liver, fibrosis, ALF), liver cirrhosis, and hepatocellular carcinoma.ALF the cause is clear, but its pathogenesis has not yet entirely clear. The hepatic stellate cells (hepatic stellate cells, HSCs) and the activation and proliferation is a key link of liver fibrosis. In recent more than 10 years at home and abroad research showed that hypoxia inducible factor -1 (hypoxia inducible factor-1, HIF-1) as a housekeeping gene regulation of hypoxia response. Through its target genes involved in regulation of HSCs activation and proliferation of.HIF-1 is composed of the heterologous regulatory subunit of HIF-1 alpha and HIF-1 beta two structural subunit dimer, HIF-1 alpha transcription of HIF-1 The activity factor has great influence, is regulated by oxygen dependent and non oxygen dependent signaling events in the cell level. Researchers cultivate human hepatic stellate cell line with endotoxin, found high expression of HIF-1 alpha, and signs of type I collagen activated by HSCs (Collagen I), alpha smooth muscle actin (alpha -smooth muscle actin, a -SMA) high expression, inhibited HIF-1 expression, type I collagen and alpha -SMA expression decreases. We previously observed in ALD rat model of alcohol in the building with the prolonging of the molding time, increased HIF-1 alpha mRNA and protein expression in liver tissue, suggesting that HIF-1 may involved in the pathogenesis of ALD. This study selected 200 mol/L acetaldehyde stimulated rat HSC-T6 cells, specific inhibition of HIF-1 expression, observe the HSC-T6 proliferation and -SMA expression of I alpha and Collagen. In order to clarify the cellular level expression of anti HIF-1 alpha Effect on HSC-T6 cell proliferation and -SMA expression of I alpha and Collagen, to investigate the effect of HIF-1 alpha regulation of HSCs in ALF. Methods: HSC-T6 cells were recovered with 10% fetal bovine serum, cultured for 1% mycillin high glucose DMEM medium at 5%CO2,37 deg.c cultured to the logarithmic growth phase. The experiment was divided into normal control group (the conventional culture), acetaldehyde stimulation group (final concentration 200 mol/L), group siRNA (Si RNA100nm/L+ HIF-1 alpha acetaldehyde at concentration of 200 u mol/L), HIF-1 siRNA (negative control alpha negative control group (NC, NC) siRNA+ acetaldehyde at concentration of 200 u mol/L). Using 200 mol/L acetaldehyde stimulation in rats HSC-T6 cells, HIF-1 Si RNA NC siRNA alpha, lipofectaminetm2000 by liposome transfection into HSC-T6 cells. After 6h, the replacement for the fresh complete medium to culture 48h, CCK-8 was used to detect the proliferation of rat HSC-T6 cells; cells were collected by RT-qPCR and Westernblot, respectively. Detection of HIF-1 alpha, alpha -sma and collagenimrna and protein expression level; with 4% paraformaldehyde fixed for immunofluorescence staining, the expression of -sma protein was observed. Results: 1 transfection of HIF-1 alpha siRNA alpha gene knock on effect of low HIF-1 on the proliferation of rat HSC-T6 cells transiently transfected with HIF-1 alpha sirna48h, cell growth significantly slow down, the results of CCK-8 assay: compared with normal control group, acetaldehyde stimulation group od increased obviously, the difference was statistically significant (P0.01), suggesting that 200 mol/l acetaldehyde can obviously promote the proliferation of HSC-T6 cells; the HIF-1 alpha siRNA group od compared with group B aldehyde stimulation, cell growth was significantly slowed down, and the difference was significant the difference (P0.01); HIF-1 alpha sirnanc group and acetaldehyde stimulation group, no significant difference between OD values (P 0.05).2 was detected by RT-qPCR HIF-1 alpha, alpha -sma and collagen gene expression relative to the reference gene beta -action are: The control group of HIF-1 alpha HSC-T6 cells, the expression of alpha -sma and collagen I were less; acetaldehyde stimulation group compared with normal control group, the expression of alpha HIF-1 alpha, -sma and collagen were increased (P < 0.01); knockdown of the HIF-1 gene, HIF-1 alpha siRNA group and acetaldehyde stimulation group, alpha HIF-1 the expression of -sma and collagen decrease while the alpha 1 expression decreased obviously (P < 0.01); HIF-1 alpha sirnanc group and acetaldehyde stimulation group, the expression changes of each index, there were no significant differences (P 0.05).3 was detected by Westernblot HIF-1 alpha siRNA transfected HSC-T6 cells in HIF-1 and alpha, alpha -sma collagen 1 protein expression level: Acetaldehyde stimulated HIF-1 expression of alpha, alpha -sma and collagen 1 protein content increased significantly, there was statistical significance of acetaldehyde stimulation group compared with the normal control group (P < 0.01); transfection of HIF-1 alpha sirna48h, reduce the expression of HIF-1 protein, and acetaldehyde stimulation There was statistically significant difference (P0.01), and the expression of alpha -sma and collagen 1 protein was also decreased; compared with HIF-1 group and alpha sirnanc stimulated by acetaldehyde group, the expression of each index, there were no significant differences (P 0.05) of alpha -sma protein expression in rat HSC-T6 cells observed in.4 immunofluorescence staining method that can be observed in the normal control group cells expressed a small amount of alpha -sma increased; acetaldehyde treated cells -sma protein expression; transfection of HIF-1 alpha sirna48h, the expression of -sma protein decreased. Conclusion: acetaldehyde can promote the proliferation of hepatic stellate cell activation, proliferation at low HIF-1 alpha on hepatic stellate cell inhibition effect of activation may be a potential target for the treatment of alcoholic liver fibrosis.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575

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相關(guān)期刊論文 前1條

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本文編號：1448028