Effect Of CTGF ShRNA And TIMP-1 ShRNA Cotransfection On Expressions Of CTGF, TIMP-1, Procol-α1 MRNA And Extra-cellular Matrix Secretion In Rat Hepatic Stellate Cells

Abstract:Objective:To investigate the effect of CTGF short hairpin RNA(shRNA)and TIMP-lshRNA co-transfection on the expression of TIMP-1,CTGF, typeⅢpro-collagen mRNA and extracellular matrix secretion in HSC-T6.Methods:Two recombinant plasmids, psiRNA-GFP-CTGF and psiRNA-GFP-CTGF containing EGFP successfully screened and constructed, were transfected alone or co-transfected into TGF-β1 stimulated HSC-T6 with the transfection reagent Lipofectamine 2000.There were three HSC-T6 groups: one group with vacant vector plasmids, another group with only Lipofectamine 2000,the last group with untreated, and 6 wells in each group.The cell transfection efficiency was analyzed by flow cytometry. The total RNA was obtained from each group after 36h and the expression of CTGF, TIMP-1, procol-al mRNA were assayed by reverse transcription and real time PCR. The content of procollagen typeⅢ,hyaluronate (HA),laminin (LN) in the supernatants were deternined by radio immunoassay. Results:Compared with those of the blank control, there was no difference in the expression of genes and extracellular matrix secretion in HSC-T6 in the group with vacant vector plasmids and the group with only Lipofectamine 2000.However, expression of CTGF, TIMP-1,procol-α1 mRNA were markedly down-regulated in CTGFshRNA and TIMP-1shRNA transfected HSC-T6.The contents of procollagen typeⅢ, hyaluronate(HA), laminin(LN) in the supernatants decreased significantly in CTGF shRNA and TIMP-1shRNA transfected HSC-T6 (P<0.01 or P<0.05).Moreover, the co-transfection can provoke a more decrease in the synthesis of precollagen typeⅢ, HA and LN,and expression of procol-α1 mRNA as well (P<0.01or P<0.05).Conclusions:1. CTGF shRNA and TIMP-1shRNA can down-regulate the gene expression of CTGF, TIMP-1,procol-α1 mRNA inhibit the extra-cellular matrix secretion in HSC-T6,and double interference may have the better inhibitory effect than single interference.2.So it may be more useful to anti-liver fibrosis therapy. The results encourage the further development of gene therapy for anti-liver fibrosis.