【作者】 王磊；

【导师】 李彤； 向宽辉；

【作者基本信息】 北京大学医学部 ， 病原生物学， 2022， 硕士

【摘要】 肝纤维化进展为肝硬化的风险高,其发生发展的关键因素是肝星状细胞活化。肝星状细胞活化后会高表达Ⅰ型胶原蛋白α1肽链(collagenⅠα1 peptide chain,COL1A1)和α-肌动蛋白-2(alpha-actin-2,ACTA2)等活化相关基因。本研究(1)旨在建立一个评价人肝星状细胞中COL1A1或ACTA2基因启动子活性的可视化报告系统,以此判断细胞活化状态,为抗肝纤维化药物的研究提供新的细胞模型。构建以不同长度COL1A1或ACTA2启动子序列调控增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因表达的数种重组质粒。先通过293T细胞瞬时转染实验确证EGFP表达,再用p LVX-1094-COL1A1-EGFP和p LVX-1601-ACTA2-EGFP质粒分别构建了稳转单克隆细胞株LX-2-CE和LX-2-AE。用荧光显微镜和Image J 1.49软件对细胞中EGFP的总荧光强度(total fluorescence intensity,TFI)和平均荧光强度(average fluorescence intensity,AFI)做半定量分析。本研究将4种中药活性成分或化合物和3种中成药用于实验。在10 ng/ml转化生长因子-β1(transforming growth factor-β1,TGF-β1)激活条件下,与TGF-β1单处理组相比,经过10μmol/L二氢丹参酮Ⅰ处理后,LX-2-CE或LX-2-AE的TFI和AFI均有下降趋势,但LX-2-CE中此两指标的变化更明显(P<0.05),适合用于后续研究。进一步研究具有抗肝纤维化作用的中药活性成分或中成药对LX-2-CE的作用,除了分析TFI和AFI,还用RT-q PCR和Western blot分别检测胞内靶基因的m RNA与蛋白水平。结果显示,在TGF-β1激活条件下,与TGF-β1组相比,LX-2-CE经5μmol/L二氢丹参酮Ⅰ处理24 h后,其胞内TFI和AFI、COL1A1和EGFP的m RNA与蛋白水平均显著降低(P<0.05);12.5μg/ml青蒿琥酯或800μg/ml扶正化瘀方可显著降低其胞内TFI、COL1A1的m RNA和蛋白水平以及EGFP的m RNA水平(P<0.05);800μg/ml安络化纤丸可显著降低其胞内TFI、COL1A1的m RNA和蛋白水平(P<0.05)。综上,本研究成功构建了基于COL1A1启动子调控EGFP表达的肝星状细胞活化的报告系统,可在体外直观报告活化相关标志物COL1A1表达,发现扶正化瘀方和安络化纤丸可能通过抑制活化的肝星状细胞COL1A1的表达发挥抗肝纤维化作用,为抗肝纤维化药物的筛选和研究提供了新的细胞模型。更多还原

【Abstract】 Patients with hepatic fibrosis have a high risk of progressing to liver cirrhosis.The key factor in development of hepatic fibrosis is the activation of hepatic stellate cells.After activation,hepatic stellate cells highly express activation-related genes such as collagen I α1peptide chain(COL1A1)and alpha-actin-2(ACTA2).The purpose of this study was to establish a visual reporter system to evaluate the promoter activity of COL1A1 or ACTA2 in human hepatic stellate cells,so as to determine the activation state of the cells,and to provide a new cell model for the study of anti-hepatic fibrosis drugs.Several recombinant plasmids were constructed,in which the enhanced green fluorescent protein(EGFP)gene expression was regulated by COL1A1 or ACTA2 promoter sequences of different lengths.The expression and fluorescence intensity of EGFP were confirmed by transient transfection of 293 T cells.Two stable monoclonal cell lines of LX-2-CE and LX-2-AE were constructed using p LVX-1094-COL1A1-EGFP or p LVX-1601-ACTA2-EGFP plasmid,respectively.The total fluorescence intensity(TFI)and average fluorescence intensity(AFI)of EGFP in cells were analyzed by the fluorescence microscope and Image J1.49 software using a semi-quantitative method.In this study,four active ingredients of traditional Chinese medicines or compounds and three Chinese patent medicines were used for the experiments.Under the condition of transforming growth factor-β1(TGF-β1)-induced activation,the TFI and AFI of LX-2-CE or LX-2-AE both decreased after 10 μmol/L dihydrotanshinone I treatment compared with those in the TGF-β1 alone treatment group.However,the changes of two indicators in LX-2-CE were more obvious than those in LX-2-AE(P < 0.05),suggesting that LX-2-CE was a better candidate for follow-up studies.The effect of active ingredients of traditional Chinese medicines or Chinese patent medicines on LX-2-CE was futhur studied.The selected drugs are known to have experimental and/or clinical potential against liver fibrosis.Besides analyzing the TFI and AFI in LX-2-CE,the intracellular m RNA and protein levels of target genes were detected by RT-q PCR and Western blot,respectively.Further experiments found that under the condition of TGF-β1 activation,the LX-2-CE cells treated with 5 μmol/L dihydrotanshinone I for 24 h showed significant decreases of intracellular TFI and AFI,m RNA and protein levels of COL1A1 and EGFP compared with those in the TGF-β1 group,respectively(P < 0.05).12.5 μg/ml Artesunate or800 μg/ml Fuzheng Huayu fang(FZHY)could significantly reduce the intracellular TFI,COL1A1 m RNA and protein levels and EGFP m RNA levels(P < 0.05).800 μg/ml Anluo Huaxian wan(ALHX)could significantly reduce the intracellular TFI,COL1A1 m RNA and protein levels(P < 0.05).In summary,a reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP is successfully constructed,which could visually report the expression changes of activation-related marker COL1A1 in vitro.FZHY and ALHX may play the roles in anti-hepatic fibrosis by inhibiting the expression of COL1A1 in the activated hepatic stellate cells.It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.更多还原