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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 observe the effect of lentivirus on the expression of interleukin-6 (IL-6) in rats infected with small interfering RNA (siRNA) targeting tissue inhibitor of metalloproteinase-1 (TIMP-1) Inhibition of TIMP-1 expression by hepatic stellate cell line HSC-T6. Methods Two different short segments (nt161 ~ 181 and nt445 ~ 463) of rat TIMP-1 mRNA gene sequence were selected and constructed into short hairpin siRNA1 and siRNA2 expression vector in vitro and then packaged into recombinant Lenti / siRNA1-TIMP 1 / GFP and Lenti / siRNA2-TIMP-1 / GFP, and the negative control Lenti / GFP (empty vector group) was packaged at the same time, and the HSC-T6 rat hepatic stellate cell line was infected with the titer of MOI = The infection efficiency of the virus was detected by flow cytometry and fluorescence microscopy. Western blotting was used to detect the expression of TIMP-1 protein at 7 and 9 days after infection respectively. Results HSC-T6 infection did not change the cell morphology and proliferation rate. Flow cytometry and fluorescence microscopy confirmed the infection efficiency were: empty vector group 68.23%, siRNA1 infection group 57.93%, siRNA2 infection group 51.2%, compared with normal cells, 7d and 9d after infection, siRNA1, siRNA2 infection group TIMP 1 protein expression decreased, but only siRNA1 infection group 9 d after infection significantly inhibited TIMP-1 expression, the difference was statistically significant (P <0.05). Conclusion The constructed recombinant Lenti / siRNA-TIMP-1 / GFP can effectively inhibit the expression of HSC-T6TIMP-1 protein in rat hepatic stellate cell line in a short term.