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Journal of Peking University (Health Sciences) ›› 2023, Vol. 55 ›› Issue (5): 876-885. doi: 10.19723/j.issn.1671-167X.2023.05.016

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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

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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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

CLC Number: 

  • R575.2

Figure 1

The construction process of the recombinant plasmid pLVX-COL1A1-EGFP and the principle of the reporting system A, the construction process of the recombinant plasmid pLVX-COL1A1-EGFP; B, the establishment and application of the reporter system."

Table 1

Primers used in this study"

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

Figure 2

Validation results of recombinant plasmids pWPI-COL1A1-EGFP and pLVX-COL1A1-EGFP A, agarose gel electrophoresis diagram of DNA molecular marker and plasmid pWPI-COL1A1-EGFP after PacⅠ and Bst BⅠ digestion; B, the sequencing electropherogram showed that Kozak sequence was successfully inserted into pWPI-COL1A1-EGFP; C, the sequencing electropherogram showed that Kozak sequence was successfully inserted into pLVX-COL1A1-EGFP."

Figure 3

TFI of five LX-2 monoclonal cell lines stably transfected with pLVX-COL1A1-EGFP ($\bar x \pm s$, n=3) A, five LX-2 monoclonal cell lines were treated with 10 μg/L TGF-β1 for 24 h, and photographed under the fluorescence microscope; B, the TFI of fluorescence image was analyzed by ImageJ 1.49. Take number 1 monoclonal cell line as control, the relative TFI was calculated. *P < 0.001 vs. control. ▲P < 0.001 vs. number 3."

Figure 4

The key steps in the standard operation procedure (SOP) of semi-quantitative analysis of fluorescence intensity based on ImageJ 1.49 software A, the fluorescence image yet to be analyzed; B, the gray value distribution diagram of the fluorescence image after the color format was converted into a gray format; C, the selected area of the same fluorescence image in different threshold ranges; D, separation of the connected nuclei for cell nucleus counting. The red arrows illustrated some separation lines."

Figure 5

Comparison of changes in TFI and AFI of LX-2-CE induced by dihydrotanshinone Ⅰ and artesunate at different concentrations A, LX-2-CE was treated with different concentrations of dihydrotanshinone Ⅰ/artesunate and 10 μg/L TGF-β1 for 24 h, and photographed under the fluorescence microscope; B, relative TFI and AFI of each group were calculated. L DH Ⅰ, M DH Ⅰ and H DH Ⅰ stand for 1 μmol/L, 5 μmol/L and 10 μmol/L dihydrotanshinone Ⅰ respectively. L Art, M Art and H Art stand for 12.5 mg/L, 25 mg/L and 50 mg/L artesunate respectively. Art, artesunate. DH Ⅰ, dihydrotanshinone Ⅰ; L, low; M, medium; H, high. *P < 0.001 vs. control. #P < 0.05, ★P < 0.01, ▲P < 0.001 vs. TGF-β1. $\bar x \pm s$, control n=21, TGF-β1 n=21, L DH Ⅰ n=14, M DH Ⅰ n=14, H DH Ⅰ n=21, L Art n=14, M Art n=21, H Art n=14."

Figure 6

Comparison of the changes of mRNA levels of COL1A1 and EGFP in LX-2-CE induced by different concentrations of dihydrotanshinone Ⅰ and artesunate LX-2-CE was treated with different concentrations of dihydrotanshinone Ⅰ/artesunate and 10 mg/L TGF-β1 for 24 h, the mRNA levels of COL1A1 and EGFP were measured by RT-qPCR. The mRNA levels of COL1A1 and EGFP were normalized to GAPDH. L DH Ⅰ, M DH Ⅰ and H DH Ⅰ stand for 1 μmol/L, 5 μmol/L and 10 μmol/L dihydrotanshinone Ⅰ respectively. L Art, M Art and H Art stand for 12.5 mg/L, 25 mg/L and 50 mg/L artesunate respectively. Art, artesunate. DH Ⅰ, dihydrotanshinone Ⅰ; L, low; M, medium; H, high. *P < 0.001 vs. control. ★P < 0.01, ▲P < 0.001 vs. TGF-β1. $\bar x \pm s$, Control n=12, TGF-β1 n=12, L DH Ⅰ n=10, M DH Ⅰ n=11, H DH Ⅰ n=10, L Art n=11, M Art n=11, H Art n=11."

Figure 7

Comparison of the changes of protein levels of COL1A1 and EGFP in LX-2-CE induced by different concentrations of dihydrotanshinone Ⅰ and artesunate LX-2-CE was treated with different concentrations of dihydrotanshinone Ⅰ/artesunate and 10 mg/L TGF-β1 for 24 h, the protein expression of COL1A1 and EGFP were determined by Western blot. L DH Ⅰ, M DH Ⅰ and H DH Ⅰ stand for 1 μmol/L, 5 μmol/L and 10 μmol/L dihydrotanshinone Ⅰ respectively. L Art, M Art and H Art stand for 12.5 mg/L, 25 mg/L and 50 mg/L artesunate respectively. Art, artesunate. DH Ⅰ, dihydrotanshinone Ⅰ; L, low; M, medium; H, high. *P < 0.001 vs. control. #P < 0.05, ★P < 0.01, ▲P < 0.001 vs. TGF-β1. $\bar x \pm s$, n=3."

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