关键词: 小干扰RNA, 金属蛋白酶组织抑制因子-1, 慢病毒, 肝星状细胞

Abstract: Objective To construct recombinant lentivirus carrying siRNA of TIMP-1 and to investigate the short-term inhibitory effect of TIMP-1 gene expression on rat hepatic stellate cell in vitro. Methods Two different short fragments were selected in accordance with rat TIMP-1 mRNA gene sequence, and U6 promoter followed by annealing siRNA which had the strongest suppressive effect were cloned into the lentivirus vector(PGCL/siRNA-TIMP-1/GFP) and packed in 293 cells to construct the recombinant Lenti/siRNA-TIMP-1/GFP. Experiments were divided into four groups: Lenti/siRNA1-TIMP-1/GFP, Lenti/siRNA2-TIMP-1/GFP, Lenti/GFP group and mock treatment group. Rat HSC-T6 cells were infected with these recombinant lenti-viruses at a concentration of MOI by 10. To monitor the efficiency of infection, fluorescence microscope and flow cytometer were used. After 7 and 9 days post-infection, Western blot was used to detect the TIMP-1 protein level. Results HSC-T6 had no significant changes after infection. The results of PCR restrictive enzyme digestion and gene sequencing confirmed that the PGCL/siRNA-TIMP-1 was constructed successfully. The efficiency of infection was over 50% in three groups. The protein expression levels of TIMP-1 in HSC-T6 cells at 9 day post-infection by the recombinant lentivirus were suppressed dramatically compared with those in mock treatment control and normal HSC-T6 cells(P＜0.05). Conclusion Recombinant Lenti/siRNA-TIMP-1/GFP could suppress the expression of TIMP-1 in rat HSC-T6 cells for short term effectively.

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