Objective: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs.
Methods: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot.
Results: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05).
Conclusion: A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.
目的: 建立评价人肝星状细胞中Ⅰ型胶原蛋白Ⅰα1肽链(collagen Ⅰα1 chain，COL1A1)基因启动子活性的可视化报告系统，判断细胞的活化状态，为抗肝纤维化药物的研究提供细胞模型。
方法: 以人肝癌细胞株HepG2基因组DNA为模板，扩增COL1A1启动子序列。在pLVX-AcGFP1-N1质粒基础上构建以COL1A1启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein，EGFP)基因表达的重组质粒pLVX-COL1A1-EGFP。使用慢病毒包装系统将pLVX-COL1A1-EGFP稳定转染至永生化的人肝星状细胞LX-2中，并筛选单克隆细胞株。对该细胞株使用转化生长因子-β1(transforming growth factor-β1，TGF-β1)激活及2种具有潜在抗肝纤维化作用的药物处理。使用荧光显微镜及ImageJ 1.49软件对细胞中EGFP荧光强度进行半定量分析，再分别使用逆转录实时定量PCR(reverse transcription real-time quantitative PCR，RT-qPCR)和免疫印迹试验检测胞内COL1A1和EGFP的mRNA水平与蛋白质水平。
结果: 构建了由COL1A1启动子调控EGFP表达的重组慢病毒质粒pLVX-COL1A1-EGFP，并加入了Kozak序列以增强EGFP的表达，获得了稳定转染pLVX-COL1A1-EGFP的LX-2单克隆细胞株LX-2-CE。LX-2-CE经过TGF-β1和具有潜在抗肝纤维化作用的5 μmol/L二氢丹参酮Ⅰ共处理24 h后，其总荧光强度和平均荧光强度均低于TGF-β1单处理组(P < 0.05)；胞内COL1A1和EGFP的mRNA水平与蛋白质水平同样均低于TGF-β1单处理组(P < 0.05)。
结论: 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，可在体外直观报告肝星状细胞活化相关标志物COL1A1表达，为抗肝纤维化药物的筛选和研究提供了新的细胞模型。
Keywords: Collagen Ⅰ α1 chain; Enhanced green fluorescent protein; Hepatic fibrosis; LX-2 cells; Reporter system.