以腺相关病毒为载体的小干扰RNA对肝星状细胞中金属蛋白酶组织抑制因子的抑制作用

Suppression of tissue inhibitor of metalloproteinase-1(TIMP-1) gene expression by recombinant adeno-associated virus carrying small interfering RNA(siRNA) of TIMP-1 in rat hepatic stellate cell(HSC)

目的观察以腺相关病毒(AAV)为载体含有针对大鼠金属蛋白酶组织抑制因子(TIMP)-1具有较强抑制作用的小干扰RNA(siRNA)感染大鼠星状细胞系HSC-T6后TIMP-1的表达抑制作用。方法针对大鼠TIMP-1 mRNA基因序列挑选一段22bp片段,在体外构建为短发夹siRNA (shon hairpin siRNA,shRNA)表达载体后,将其包装为重组AAV并感染大鼠肝星状细胞系HSC-T6后,于感染后30d及90d应用荧光定量PCR方法及Western blot方法分别检测TIMP-1 mRNA及蛋白质表达情况,同时通过PCR技术以感染后细胞的基因组DNA为模板扩增外源基因验证其长效表达。结果经PCR、酶切及序列测定证实含有siRNA-TIMP-1基因的重组AAV载体质粒已成功克隆。将重组质粒包装成病毒后感染HSC-T6细胞,与对照组细胞相比,感染后30d及90d细胞TIMP-1 mRNA水平明显降低(P＜0．01),感染后30d TIMP-1蛋白表达水平较对照组细胞相比下降约60％,而感染后90d,TIMP-1蛋白表达几乎下降90％。PCR结果显示在重组病毒感染后90d细胞基因组DNA中仍可扩增出外源基因,证实外源基因可长期表达。结论重组病毒rAAV/siPtNA-TIMP-1/neo可长期有效地抑制TIMP-1基因的表达。

Objective To construct recombinant adeno-associated virus（AAV） carrying siRNA of TIMP- 1 and to investigate the long-term effect of TIMP-1 gene RNA interference on rat hepatic steUate cell（HSC）-T6 cells in vitro. Methods U6 promoter together with the following annealing siRNA which had the strongest suppression effect were cloned into the AAV vector（pd16-95/siRNA-TIMP-1/neo） and packed in 293 cells to construct the recombinant AAV/siRNA-TIMP-1/nco. Rat HSC-T6 cells were infected by this recombinant AAV. After G418 selection, real-time fluorescence quantitative PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in HSC-T6 cells at 30 d and 90 d post-infection. At the same time, PCR was performed using the DNA of the infected cells as template to amplify U6 promoter. Results The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pd16-95/siRNA- TIMP-1/neo was constructed successfully. The transcription and expression level of TIMP-1 in HSC-T6 cells infected by the recombinant AAV were suppressed dramatically compared with those in mock control and normal HSC-T6 cells（ P 〈 0.01 ）, and the expression level of TIMP-1 protein in HSC-T6 cells decreased by 90% at 90 d and 60% at 30 d post-infection. The amplified rat U6 promoter by PCR using the DNA of infected cells as template proved that the HSG-T6 cells could continually express siRNA-TIMP-1 for 90 d after infection. Conclusion Recombinant rAAV/siRNA-TIMP-1/nco could effectively suppress the expression of TIMP-1 gene for a long time.