慢病毒为载体的siRNA抑制肝星状细胞TIMP-1的表达

Suppression of Tissue Inhibitor of Metalloproteinase-1 Gene Expression by Recombinant Lentivirus Carrying Small Interfering RNA of TIMP-1 in Rat Hepatic Stellate Cell

目的观察以慢病毒为载体,用含有针对大鼠金属蛋白酶组织抑制因子(tissue inhibitor of metalloproteinase-1,TIMP-1)、具有较强抑制作用的小干扰RNA(small interfering RNA,siRNA)感染大鼠肝星状细胞系HSC-T6后对TIMP-1表达的抑制作用。方法针对大鼠TIMP-1 mRNA基因序列挑选2个不同短片段(nt161～181和nt445～463),在体外构建为短发夹siRNA1、siRNA2表达载体后,将其包装为重组Lenti/siRNA1-TIMP-1/GFP和Lenti/siRNA2-TIMP-1/GFP,同时包装阴性对照Lenti/GFP(空载体组),并以滴度MOI=10感染大鼠肝星状细胞系HSC-T6,感染后3d,用流式细胞仪及荧光显微镜检测病毒的感染效率。分别在感染后7、9d用Western blotting方法检测TIMP-1蛋白表达情况。结果感染HSC-T6后细胞形态和增生速度未发生明显变化。流式细胞仪及荧光显微镜检查证实感染效率分别为:空载体组68.23%,siRNA1感染组57.93%,siRNA2感染组51.2%,与正常细胞相比,感染后7d和9d,siRNA1、siRNA2感染组TIMP-1蛋白表达均有所下降,但只有siRNA1感染组在感染后9d能显著抑制TIMP-1表达,差异有统计学意义(P<0.05)。结论构建的重组Lenti/siRNA-TIMP-1/GFP可短期有效抑制大鼠肝星状细胞系HSC-T6TIMP-1蛋白表达。

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/siRNAI-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.