【作者】 刘加群；

【导师】 张建军；

【作者基本信息】 泸州医学院 ， 内科学， 2009， 硕士

【摘要】 目的:结缔组织生长因子(CTGF)是新近发现的一种促肝纤维化的重要细胞因子,是转化生长因子β(transforming growth factorβ,TGF-β)信号传导途径的下游效应介质。CTGF可直接介导原代肝星状细胞(hepatic stellate cell,HSC)活化、增殖及迁移,还能促进活化HSC的合成及分泌细胞外基质(extracellular matrix,ECM)。金属蛋白酶组织抑制因子1(TIMP-1)也是TGF-β信号传导途径的下游效应介质,在肝纤维化发生时,它主要由激活的HSC表达。TIMP-1不仅抑制基质金属蛋白酶(matrix metalloproteinase, MMPs)活性阻止胶原降解,也通过抑制HSC凋亡、促进胶原分泌而在肝纤维化病情进展中发挥关键作用。所以,减少CTGF和TIMP-1的表达,可以减轻肝纤维化程度。本研究旨在构建以大鼠CTGF或TIMP-1基因为靶点的短发夹RNA(short hairpin RNA, shRNA)表达载体,为进一步探索肝纤维化的基因治疗提供有力工具。方法:根据前期筛选出的对CTGF和TIMP-1基因最有效的RNA干扰靶位,即CTGF基因1560～1580nt和TIMP-1基因412～432nt ,按照RNA干扰(RNA interference,RNAi)序列设计原则,各设计1对含有短发夹结构的RNAi靶点序列,退火形成双链DNA,分别克隆到经双酶切后的质粒载体psiRNA-h7SKGFPzeo,构建成含目的靶基因片段的重组质粒载体psiRNA-GFP-CTGF和psiRNA-GFP-TIMP-1,并对重组质粒进行双酶切琼脂糖电泳和测序鉴定。结果:酶切证实以大鼠CTGF或TIMP-1基因为靶点的表达shRNA的目的基因片段分别被成功的克隆到质粒载体psiRNA-h7SKGFPzeo中,测序结果证明重组质粒载体的插入序列与设计的靶基因片段完全一致。结论:成功构建靶向大鼠CTGF和TIMP-1最有效的RNA干扰靶位的shRNA表达质粒重组体,为进一步探索肝纤维化基因治疗的新途径打下了实验基础。更多还原

【Abstract】 objective: Connective tissue growth factor (CTGF) is a recently discovered important cytokine of promoting hepatic fibrosis,which is transforming growth factor-β(TGF-β) signal transduction pathway downstream effect factor, which not only can directly mediate primary hepatic stellate cell (HSC) activation, proliferation and migration, but also can promote the HSC to synthesize and secret extracellular matrix. Another effect factor of TGF-βsignal transduction pathway downstream, Tissue inhibitor of metalloproteinase 1 (TIMP-1) is mainly expressed by activated HSC during the progress of hepatic fibrosis, which not only inhibits matrix metalloproteinase (matrix metalloproteinase, MMPs) activity to prevent collagen degradation, but also inhibits HSC apoptosis and promote collagen secreting. In the progress of hepatic fibrosis disease, TIMP-1 plays a key role. Therefore, reduced CTGF and TIMP-1 expression maybe could lessen hepatic fibrosis. The purpose of this study is to construct shRNA (short hairpin RNA) expression plasmid vectors targeting to rat connective tissue growth factor (CTGF) or tissue inhibitor of metalloproteinase 1 (TIMP-1). This study provides a powerful tool for further exploring a new gene therapy way of hepatic fibrosis in the future. Methods: According to the RNA interference sequences of rat CTGF gene (1560～1580nt) and rat TIMP-1 (412～432nt) , the targeted sequences had been screened out in the primary experiments, and based on the RNA interference (RNAi) sequence of design principles, two pairs of oligonucleotides were synthesized chemically, and then subjected to be annealed to form two double-stranded DNA fragments, respectively. The two double-stranded DNA fragments were then cloned into the plasmid vector, psiRNA-h7SKGFPzeo, respectively. The recombinant plasmids were named as psiRNA-GFP-CTGF or psiRNA-GFP-TIMP-1, which shoμld product shRNA in eukaryocytes. The recombinant plasmids psiRNA-GFP-CTGF or psiRNA-GFP-TIMP-1 was verified by Agarose gel electrophoresis and sequencing. Resμlts:By Agarose gel electrophoresis and sequencing, the two double-stranded DNA fragments were inserted correctly in psiRNA-h7SKGFPzeo vectors as expected, respectively. Conclusions: Two recombinant plasmids, psiRNA-GFP-CTGF and psiRNA-GFP-CTGF were successfμlly constructed. These new plasmids targeting to the rat CTGF and TIMP-1 for RNA interference shRNA expression woμld contribute to further explore new ways of gene therapy experiments for hepatic fibrosis.更多还原