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目的:探讨靶向结缔组织生长因子(CTGF)的锤头核酶抑制TGF-β1作用下人肝星状细胞(HSC)Ⅰ型胶原(Col I)合成及其细胞周期进程的作用.方法:构建含有人CTGF锤头核酶cDNA序列的重组质粒pTriCTGF-Rz.将空质粒pTriEx2和重组质粒pTriCTGF-Rz分别转染人肝星状细胞系(LX-2)细胞.细胞分为4组:pTriEx2转染组,pTriEx2转染加TGF-β1组,pTriCTGF-Rz转染加TGF-β1组和pTriCTGF-Rz转染组.采用半定量RT-PCR测定LX-2细胞CTGF mRNA和Col I mRNA转录水平,采用ELISA和流式细胞仪分别用于LX-2细胞Col I分泌功能和LX-2细胞周期进程的检测.结果:TGF-β1可明显提高LX-2细胞CTGFmRNA和Col I mRNA的转录水平及分泌ColI蛋白功能(t=11.14,14.36,7.17,均P<0.01);p Tr i C T G F-R z转染L X-2细胞既能降低基础CTGF mRNA和Col I mRNA水平及Col I蛋白水平(t=2.86,3.06,2.97,均P<0.05),又能部分拮抗TGF-β1诱导LX-2细胞CTGF mRNA和Col I mRNA转录和Col I蛋白分泌的增加(t=2.99,3.09,3.02,均P<0.05).TGF-β1对LX-2细胞周期进程无影响.结论:CTGF是TGF-β1作用下人肝星状细胞合成Col I的下游介导者,TGF-β1对HSC周期进程无影响,靶向CTGF有可能成为肝纤维化基因治疗的新靶点.
Objective: To investigate the effect of hammerhead ribozymes targeting connective tissue growth factor (CTGF) on the synthesis and cell cycle progression of type Ⅰ collagen (Col I) of human hepatic stellate cells (HSC) under the action of TGF-β1.Methods: Recombinant plasmid pTriCTGF-Rz containing the human CTGF hammerhead ribozyme cDNA sequence was transfected into human hepatic stellate cell line (LX-2) by using empty plasmid pTriEx2 and recombinant plasmid pTriCTGF-Rz respectively.The cells were divided into 4 groups: pTriEx2 The expression of CTGF mRNA and Col I mRNA in LX-2 cells was detected by semi-quantitative RT-PCR, and transfected with pTriCTGF-Rz plus TGF-β1 and pTriCTGF- ELISA and flow cytometry were used to detect the secretion of Col I and the cycle progression of LX-2 cells in LX-2 cells.Results: TGF-β1 significantly increased the mRNA and protein levels of CTGF mRNA and Col I in LX-2 cells The expression of CTI mRNA, Col I mRNA and Col I protein in transfected LX-2 cells by pTT CTG FRz (t = 11.14, 14.36, 7.17, all P <0.01) 2.86,3.06,2.97, all P <0.05), but also partially antagonized the increase of CTGF mRNA and Col I mRNA transcription and Col I protein secretion in LX-2 cells induced by TGF-β1 (t = 2.99, 3.09, 3.02 , All P <0.05) .TGF-β1 had no effect on the cell cycle progression of LX-2 cells.Conclusion: CTGF is a downstream mediator of Col I synthesis by human hepatic stellate cells under the action of TGF-β1. TGF- No effect, targeting CTGF may become a new target for gene therapy of liver fibrosis.