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北京大学学报（医学版） ›› 2023, Vol. 55 ›› Issue (5): 876-885. doi: 10.19723/j.issn.1671-167X.2023.05.016

• 论著 • 上一篇    下一篇

基于COL1A1启动子和增强型绿色荧光蛋白基因建立人肝星状细胞活化的细胞模型

王磊,金香淑,董慧君,欧国敏,赖鑫源,庄辉,李彤*(),向宽辉*()   

  1. 北京大学基础医学院病原生物学系和感染病研究中心, 北京 100191
  • 收稿日期:2021-12-21 出版日期:2023-10-18 发布日期:2023-10-09
  • 通讯作者: 李彤,向宽辉 E-mail:toglii97@bjmu.edu.cn;kxiang@bjmu.edu.cn
  • 基金资助:
    国家"十三五"艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2017ZX10202202-004-004);国家自然科学基金项目(81873579)

Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein

Lei WANG,Xiang-shu JIN,Hui-jun DONG,Guo-min OU,Xin-yuan LAI,Hui ZHUANG,Tong LI*(),Kuan-hui XIANG*()   

  1. Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
  • Received:2021-12-21 Online:2023-10-18 Published:2023-10-09
  • Contact: Tong LI,Kuan-hui XIANG E-mail:toglii97@bjmu.edu.cn;kxiang@bjmu.edu.cn
  • Supported by:
    the Major Science and Technology Special Project of China Thirteenth Five-year Plan(2017ZX10202202-004-004);the National Natural Science Foundation of China(81873579)

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摘要：

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。

关键词: 肝纤维化, LX-2细胞, Ⅰ型胶原蛋白α1肽链, 增强型绿色荧光蛋白, 报告系统

Abstract:

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.

Key words: Hepatic fibrosis, LX-2 cells, Collagen Ⅰ α1 chain, Enhanced green fluorescent protein, Reporter system

中图分类号: 

  • R575.2

图1

重组质粒pLVX-COL1A1-EGFP的构建流程和报告系统原理"

表1

本研究所用引物"

Use of primers Sequence of primer (5′→3′) Product length/bp
Construction of plasmid
    Fragment 1 of overlap PCR Forward: CAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAG
Reverse: GTTTTGTTTGCCATGGCTGTGTTCTGGCGGC		 1 229
    COL1A1 promoter (Fragment 2 of overlap PCR) Forward: GCCGCCAGAACACAGCCATGGCAAACAAAAC
Reverse: ATGATGTTAATTAACATGTAGACTCTTTGTGGCTG		 1 123
    EGFP containing Kozak-sequence Forward: ATGATGTTAATTAA$\underline{{\rm{GCCACCATGG}}}$TGAGCAAGGGCGA
Reverse: ATGATGTTCGAAGCTTGAGCTCGACTAGCTA		 802
    Fragment of pLVX-COL1A1-EGFP Forward: ATGATGCAGAGATCCAGTTTATCCATGGCAAACAAAACTC
Reverse: ATGATGCGGTAGAATTATCTAGTTACTTGTACAGCTCGTCCAT		 1 872
RT-qPCR
    GAPDH Forward: AAGAAGGTGGTGAAGCAGGC
Reverse: TCCACCACCCTGTTGCTGTA		 203
    COL1A1 Forward: ATGTGCCACTCTGACTGGAA
Reverse: ACCAGTCTCCATGTTGCAGA		 99
    EGFP Forward: GCAGAAGAACGGCATCAAGG
Reverse: CGGACTGGGTGCTCAGGTAG		 149

图2

重组质粒pWPI-COL1A1-EGFP和pLVX-COL1A1-EGFP的验证结果"

图3

稳定转染pLVX-COL1A1-EGFP的5个LX-2单克隆细胞株的TFI($\bar x \pm s$, n=3)"

图4

基于ImageJ 1.49软件的荧光强度半定量分析方法的SOP中关键步骤"

图5

不同浓度的二氢丹参酮Ⅰ和青蒿琥酯诱导LX-2-CE的TFI和AFI变化的比较"

图6

不同浓度二氢丹参酮Ⅰ和青蒿琥酯诱导LX-2-CE的COL1A1和EGFP的mRNA水平变化的比较"

图7

不同浓度二氢丹参酮Ⅰ和青蒿琥酯诱导LX-2-CE的COL1A1和EGFP蛋白质水平变化的比较"

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ISSN 1671-167X
CN 11-4691/R
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