靶向CTGF锤头核酶对TGF-β1作用下人肝星状细胞合成Ⅰ型胶原的作用  

Inhibitory effect of hammerhead ribozyme targeting connective tissue growth factor on TGF-β1-induced collagen I synthesis in human hepatic stellate cells

作　　者：齐晓艳[1] 高润平[1] 王淑华[2] 张瑞娟[1] 包万国[1] 金清龙[1] 辛桂杰[1] 杨永广[3] 

机构地区：[1]吉林大学第一医院感染症科,吉林省长春市130021 [2]吉林大学第一医院激光科,吉林省长春市130021 [3]哈佛医学院马塞诸塞总院

出　　处：《世界华人消化杂志》2008年第23期2587-2591,共5页World Chinese Journal of Digestology

基　　金：吉林省科技厅资助项目;No.200505211;No.20070729-9~~

摘　　要：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.AIM： To observe the effect of hammerhead ribozyme targeting connective tissue growth factor （CTGF） on TGF-β1-induced collagen I synthesis and cell cycle progression in human hepatic stellate cells （HSCs）. METHODS： CTGF hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to construct recombinant vector pTriCTGF-Rz. Both vectors were trans- fected into human hepatic stellate cell line （LX-2） individually, which was then stimulated by addition of TGF-β1 to the culture media. Semi- quantitative reverse-transcription polymerase chain reaction was used to determine the tran- scription of CTGF mRNA and collagen I mRNA in LX-2 cells. Collagen I secretion and cell cycle progression were measured by enzyme-linked immunosorbent assay （ELISA） and flow cytom- etry, respectively. RESULTS： TGF-β1 obviously increased the tran- scription of CTGF mRNA and collagen I mRNA and secretion of collagen I protein in pTriEx2- transfected LX-2 cells （t = 11.14, 14.36, 7.17; all P 〈 0.01）. pTriCTGF-Rz-transfected LX-2 cells showed a decrease in the basic transcription of CTGF mRNA and collagen I mRNA as well as in the secretion of collagen I protein （t = 2.86, 3.06, 2.97; all P 〈 0.05）. Furthermore, TGF-β1-induced increase of CTGF mRNA and collagen I mRNA transcription as well as collagen I secretion were partially inhibited in pTriCTGF-Rz-transfected LX-2 cells （t = 2.99, 3.09, 3.02; all P 〈 0.05）. TGF-β1 had no effect on LX-2 cell cycle progres- sion. CONCLUSION： CTGF is an essential downstream mediator for TGF-β1-induced collagen I production in human HSCs, but TGF-β1 has no effect on CTGF-mediated cycle progression of HSCs. CTGF may become a new target of gene therapy for liver fibrosis.

关 键 词：结缔组织生长因子 TGF-Β1 人肝星状细胞 锤头核酶 基因治疗 逆转录-聚合酶链反应 

分 类 号：R575.2[医药卫生—消化系统] R285.5[医药卫生—内科学]