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[Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein].
Wang, Lei; Jin, Xiang Shu; Dong, Hui Jun; Ou, Guo Min; Lai, Xin Yuan; Zhuang, Hui; Li, Tong; Xiang, Kuan Hui.
Afiliación
  • Wang L; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Jin XS; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Dong HJ; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Ou GM; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Lai XY; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Zhuang H; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Li T; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
  • Xiang KH; Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
Beijing Da Xue Xue Bao Yi Xue Ban ; 55(5): 876-885, 2023 Oct 18.
Article en Zh | MEDLINE | ID: mdl-37807743
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-ß1 (TGF-ß1) or co-treated with TGF-ß1 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-ß1 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-ß1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-ß1 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.
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Texto completo: 1 Base de datos: MEDLINE Asunto principal: Factor de Crecimiento Transformador beta1 / Células Estrelladas Hepáticas Tipo de estudio: Prognostic_studies Idioma: Zh Revista: Beijing Da Xue Xue Bao Yi Xue Ban Asunto de la revista: MEDICINA Año: 2023 Tipo del documento: Article

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Factor de Crecimiento Transformador beta1 / Células Estrelladas Hepáticas Tipo de estudio: Prognostic_studies Idioma: Zh Revista: Beijing Da Xue Xue Bao Yi Xue Ban Asunto de la revista: MEDICINA Año: 2023 Tipo del documento: Article