关键词: 结缔组织生长因子, 人肝星状细胞, 基因治疗

Abstract: Abstract:Objective To study the inhibitory effect of hammerhead ribozyme targeting connective tissue growth factor (CTGF) on collagen I synthesis and cell cycle progression of human hepatic stellate cell line (LX-2) cells.Methods Hammerhead ribozyme cDNA targeting CTGF mRNA plus two self-cleaving sequences were inserted into pTriEx2 vector to construct a recombinant vector pTriCTGF-Rz.LX-2 cells were transfected with either pTriEx2 or pTriCTGF-Rz and further stimulated with or without TGF-1. There were five groups in the experiment:control group,pTriEx2 group,pTriCTGF-Rz group,pTriEx2 plus TGF-β1 group,and pTrCTGF-Rz plus TGF-β1 group.Semi-quantitative RT-PCR was used to detect the levels of CTGF mRNA and collagen Ⅰ mRNA.ELISA and flow cytometry were used to detect the levels of collagen Ⅰ secretion and cell cycle.Results Transfection of pTriCTGF-Rz into LX-2 cells reduced the CTGF mRNA and collagen Ⅰ mRNA levels as well as collagen 　Ⅰ protein level compared with pTriEx2 group(P<0.05).TGF- 1-induced CTGF mRNA and collagen Ⅰ mRNA transcription were decreased in LX-2 cells transfected with pTriCTGF-Rz compared with control groups (P<0.05).Furthermore,in the LX-2 cells transfected with pTriCTGF-Rz,the proportion of cells at G₀-G₁ phase increased (P<0.05)　 and that of cells at S phase decreased(P<0.01) compared with control group.Conclusion The hammerhead ribozyme targeting CTGF has inhibitory effects on human hepatic stellate cell-mediated fibrosis and it may become another candidate for gene therapy in the future.

Key words: connective tissue growth factor, human hepatic stellate cell, gene therapy