Effect of CTGF shRNA on expressions of TGF-β1,CTGF mRNA and extra-cellular matrix secretion in rat hepatic stellate cells

Objective: To investigate the effect of CTGF short hairpin RNA(shRNA) on the expression of TGF-β1,CTGF,type Ⅰ procollagen,type Ⅲ procollagen mRNA and extracellular matrix secretion in HSC-T6.Methods: The CTGFshRNA plasmids containing EGFP successfully constructed and screened,were transfected to HSC-T6 with the transfection reagent Metafectene.There were another three HSC-T6 groups: one with plasmids and without CTGFshRNA,one with only Metafectene,and one untreated.The expression of TGF-β1,CTGF,type Ⅰ procollagen and type Ⅲ procollagen mRNA levels were determined by reverse transcription-polymerase chain reaction(RT-PCR) after 24 and 48 hours.The content of precollagen type Ⅲ,Ⅳ-type collagen,and hyaluronate(HA), laminin(LN) in the supernatants were determined by radioimmunoassay.Cell transfection efficiency was analyzed by flow cytometry.Results: The transfection efficiency of CTGFshRNA plasmids in HSC were(60±5)%,(42±3)% at 24 and 48 hours respectively.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 other groups,the group with plasmids and without CTGF shRNA and the group with only Metafectene.Expression of CTGF,type Ⅰ collagen and type Ⅲ collagen mRNA were markedly down-regulated in CTGFshRNA transfected HSC-T6(P0.01 or P0.05).But there was no difference in the expression of TGF-β1 mRNA. The contents of precollagen type Ⅲ,Ⅳ-type collagen,hyaluronate(HA) ,and laminin(LN) in the supernatants decreased significantly in CTGFshRNA transfected HSC-T6(P0.01 or P0.05).Conclusion: CTGFshRNA can regulate the gene expression of the growth factor and the extra-cellular matrix molecule and inhibits the extra-cellular matrix secretion in HSC-T6.