敲低HIF-1α基因对乙醛刺激的肝星状细胞增殖活化影响的实验研究

发布时间：2018-01-20 11:31

本文关键词： 低氧诱导因子-1 乙醛肝星状细胞 α-平滑肌肌动蛋白酒精性肝纤维化　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:酒精性肝病(alcoholic liver disease,ALD)是因过量摄入酒精引发的中毒性肝脏病变,早期通常表现为酒精性脂肪肝(alcoholic fatty liver disease,AFLD)可进展为酒精性肝炎、酒精性肝纤维化(alcohol liver fibrosis,ALF)、肝硬化、甚至肝癌。ALF病因明确,但其发病机制尚未完全清楚。目前认为肝星状细胞(hepatic stellate cells,HSCs)增殖与活化是肝纤维化发生的核心环节。近十多年来国内外研究表明,缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)作为低氧应答调控的管家基因,可能通过其调节的靶基因参与HSCs的活化与增殖。HIF-1是由调节亚基HIF-1α与结构性亚基HIF-1β共同组成的异源二聚体,HIF-1α对HIF-1发挥转录因子活性起着至关重要的影响,在细胞内水平受到氧依赖和非氧依赖信号事件的调控。有研究者用内毒素培育人肝星状细胞系,发现HIF-1α高表达,且标志HSCs活化的I型胶原蛋白(Collagen I)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)高表达,抑制HIF-1α表达后,I型胶原蛋白及α-SMA表达也随之减低。我们前期在酒精灌胃构建的大鼠ALD模型中观察到:随着造模时间的延长,肝组织HIF-1αmRNA和蛋白表达随之增多,提示HIF-1α可能参与着ALD发病过程。本研究选用200μmol/L乙醛刺激大鼠HSC-T6细胞,特异性抑制HIF-1α表达,观察HSC-T6细胞增殖情况及α-SMA和Collagen I表达变化。旨在细胞水平阐明抑制HIF-1α表达对HSC-T6细胞增殖及α-SMA和Collagen I表达影响,探讨HIF-1α调控的HSCs在ALF中的作用。方法:复苏HSC-T6细胞用含10%胎牛血清、1%青链霉素的高糖DMEM培养基在5%CO2,37℃条件下培养到对数生长期。实验分为正常对照组(常规培养)、乙醛刺激组(终浓度为200μmol/L)、HIF-1αsiRNA组(si RNA100nm/L+乙醛终浓度为200μmol/L)、HIF-1αsiRNA阴性对照(negative control,NC)组(NC siRNA+乙醛终浓度为200μmol/L)。利用200μmol/L乙醛刺激大鼠HSC-T6细胞,将HIF-1αsi RNA、NC siRNA通过脂质体lipofectaminetm2000瞬时转染至hsc-t6细胞,培养6h后,更换为新鲜完全培养液继续培养48h,应用cck-8检测大鼠hsc-t6细胞增殖变化;收集细胞,分别利用rt-qpcr和westernblot法检测hif-1α、α-sma和collagenimrna和蛋白表达水平;用4%多聚甲醛固定,行免疫荧光染色,观察α-sma蛋白表达情况。结果:1转染hif-1αsirna敲低hif-1α基因对大鼠hsc-t6细胞增殖的影响瞬时转染hif-1αsirna48h后,细胞增长情况明显减慢,cck-8比色结果:与正常对照组比较,乙醛刺激组od值明显升高,差异具有统计学意义(p0.01),提示200μmol/l乙醛可明显促进hsc-t6细胞增殖;hif-1αsirna组od值与乙醛刺激组比较,细胞增长情况明显减慢,且差异具有统计学意义(p0.01);hif-1αsirnanc组与乙醛刺激组比较,od值差异无统计学意义(p均0.05)。2采用rt-qpcr法检测hif-1α、α-sma和collagenⅠ基因相对于β-action内参基因表达变化:正常对照组hsc-t6细胞中hif-1α、α-sma和collagenⅠ的表达均较少;乙醛刺激组与正常对照组比较,hif-1α、α-sma和collagenⅠ表达量明显增多(p均0.01);敲低hif-1α基因,hif-1αsirna组与乙醛刺激组比较,hif-1α表达降低的同时α-sma和collagenⅠ表达明显减少(p均0.01);hif-1αsirnanc组和乙醛刺激组比较,各个指标表达的变化,差异均无统计学意义(p均0.05)。3通过westernblot法检测hif-1αsirna转染后hsc-t6细胞中的hif-1α、α-sma和collagenⅠ蛋白水平表达变化:乙醛刺激组hif-1α、α-sma和collagenⅠ蛋白表达量明显增多,乙醛刺激组与正常对照组比较差异有统计学意义(p均0.01);转染hif-1αsirna48h后,hif-1α蛋白表达降低,与乙醛刺激组比较差异具有统计学意义(p0.01),且α-sma和collagenⅠ蛋白的表达量也减低;hif-1αsirnanc组与乙醛刺激组比较,各个指标表达变化,差异均无统计学意义(p均0.05)。4免疫荧光染色法观察大鼠hsc-t6细胞中α-sma蛋白表达情况:可观察到正常对照组细胞表达少量α-sma;乙醛刺激组细胞α-sma蛋白表达增多;转染hif-1αsirna48h后,细胞中的α-sma蛋白表达减少。结论:乙醛能够促进肝星状细胞增殖活化,敲低HIF-1α对肝星状细胞的增殖活化有抑制作用,可能是酒精性肝纤维化的潜在治疗靶点。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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