Construction Of Recombinant ShRNA-espressing Vectors Targeting To Two Different Hepatic Fibrosis-related Cytokines And Expression In Hepatic Stellate Cells

bjective:Connective tissue growth factor (CTGF) is a recently discovered important cytokine of promoting hepatic fibrosis, which is a transforming growth factor-P (TGF-P) signal transduction pathway downstream effect factor, and can not only directly mediate primary hepatic stellate cell (HSC) activation, proliferation and migration, but also promote the HSC to synthesize and secret extracellular matrix. Another effect factor of TGF-P signal 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) and/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 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-DUO-GFPzeo, respectively. To construct the target gene fragment containing the target vector of psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA-GFP-Com (with CTGF and TIMP-1).The and recombinant plasmids were verified by agarose gel electrophoresis and sequencing. Recovery in vitro cultured, when the rat HSCs in logarithmic growth phase cells in 0.25% trypsin grew to be about 80% confluence,3×105/ml seeded in six-well plate until the cells grew to 80% confluence, serum-free Opti-MEM medium for synchronization using liposomes (TransFast Transfmlection Reagent) 2000 mediated, and the recombinant plasmids (psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA-GFP-Com (with CTGF and TIMP-1) and the psiRNA-DUO-GFPzeo transiently transfected by liposomes (TransFast Transfection Reagent) 2000. Fluorescence microscope and flow cytometry were employed to meaure the plasmid transfection the efficiency.Results:1:confirmed by rats CTGF or TIMP-1 gene expression for the purpose of the target shRNA genetic fragments were successfully cloned by plasmid carrier psiRNA DUO-GFPzeo and sequencing result proves the carrier recombination plasmid insert sequence and design target genes segment.2: using instantaneous transfection hepatic stellate cell fluorescence microscope observation, after transfection into plasmids in all cells send green fluorescence, preliminary proven transfection into hepatic stellate cells. Conclusion:the successful construction target CTGF rats and TIMP-1 the most effective RNA interference shRNA expression plasmid sites of recombinant. Instantaneous transfection transfection to rat hepatic stellate cell fluorescence microscope, can be found in the cell, with fluorescent transfection success into hepatic stellate cells. So as to further explore gene therapy for hepatic fibrosis new ways of experimental basis laid.