关键词: 金属蛋白酶组织抑制因子-1, 小干扰RNA, 肝星状细胞

Abstract: Objective To investigate the effects of RNA interference targeting TIMP-1 gene on rat hepatic stellate cell(HSC)-T6 cells in vitro and screen highly efficient small interfering RNA(siRNA).Methods Five pair of 21 nucleotide siRNAs targeting rat TIMP-1 at nt161～181,nt 190～208,nt 208～226,nt 226～244,nt 445⁴63 and one pair of fluorescence labeled unspecific siRNA were synthesized.After transfecting different concentrations of unspecific siRNA to select the optimum transfection concentration,50 nmol/L siRNAs were transfected into rat HSC-T6 cells respectively.The protein of the infected cells at 24 h,48 h and 72 h was collected and detected by Western blot to confirm the suppression effects of diverse siRNAs at different time point.Results It was demonstrated by fluorescence actived cell sorter that 50 nmol/L siRNA had the optimum trasfection efficacy,and 3 of the 5 siRNA oligomers had the significant effects on suppressing TIMP-1 expression by Western-blot within 48 hours after transfecting into rat HSC-T6 cells.The expression level of TIMP-1 gene in HSC-T6 cells infected by one pair of siRNA after 48 h decreased significantly(80%) compared with control cells,and the effects of suppression disappeared at 72 h after transfection.Conclusion RNA interference can exert an suppression of TIMP-1 gene in rat HSC.One paie of siRNA,which can highly effectively inhibit expression of TIMP-1 gene was screened successfully,and it will be reconstructed into virus vectors to suppress the specific gene expression for a long time by chromosomal integration.

Key words: tissue inhibitor of metalloproteinase-1, small interfering RNA, hepatic stellate cell