Construction Of Recombinant ShRNA-espressing Vectors Targeting To Two Different Hepatic Fibrosis-related Cytokines

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.