靶向CTGF锤头核酶对人肝星状细胞Ⅰ型胶原及细胞周期的影响

Effects of hammerhad ribozyme targeting connective tissue growth factor on collagen Ⅰ synthesis and cell cycle progression of human hepatic stellate cells

目的:探讨靶向人结缔组织生长因子(CTGF)锤头核酶对人肝星状细胞系(LX-2)细胞Ⅰ型胶原转录和分泌及细胞增殖的抑制作用,为肝纤维化基因治疗提供实验依据。方法:构建含有CTGF锤头核酶及其5′和3′端分别连接一自剪酶cDNA序列的重组质粒pTriCTGF-Rz。实验分5组:未转染组、空质粒pTriEx2转染组、空质粒转染加TGF-1组、pTriCTGF-Rz转染组和pTriCTGF-Rz转染加TGF-1组。半定量RT-PCR检测CTGF mRNA和Ⅰ型胶原mRNA水平,ELISA检测LX-2细胞分泌Ⅰ型胶原的水平,流式细胞仪检测LX-2细胞周期。结果:与pTriEx2转染组比较,pTriCTGF-Rz组LX-2细胞CTGF mRNA水平和TGF-1诱导CTGFmRNA的水平降低(P<0.05),pTriCTGF-Rz组LX-2细胞Ⅰ型胶原mRNA转录和蛋白分泌减少,TGF-1诱导I型胶原的转录水平降低(P<0.05)。与空质粒pTriEx2转染组比较,pTriCTGF-Rz转染组LX-2细胞G0-G1期细胞数增多(P<0.05),S期细胞减少(P<0.01)。结论:靶向CTGF的锤头核酶具有抑制人肝星状细胞介导肝纤维化的作用,可能成为肝纤维化基因治疗的新途径。

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 steliate cell line （LX-2） ceils. 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.