腺相关病毒介导的小干扰RNA对大鼠肝星状细胞金属蛋白酶组织抑制因子-1及基质金属蛋白酶13表达的影响

Expression of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase 13 in rat hepatic stellate cell infected by adeno-associated virus carrying small interfering RNA

目的观察以腺相关病毒(AAV)为载体含有针对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及基质金属蛋白酶13(MMP13)的表达情况。方法将对大鼠TIMP-1基因具有最强抑制作用的一对siRNA,在体外构建为短发夹siRNA表达载体后,将其包装为重组AAV-rAAV/siRNA-TIMP-1/neo并感染HSC-T6,于感染后4周及12周应用荧光定量PCR方法及Western blot方法分别检测TIMP-1及MMP13mRNA及蛋白质表达情况。结果经PCR、酶切及序列测定证实抑制作用最强的1对siRNA在体外构建的shRNA表达载体成功克隆。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,rAAV/siRNA-TIMP-1/neo感染组在感染后4周及12周细胞TIMP-1mRNA及蛋白质表达水平明显降低(P<0.01),而MMP13mRNA及蛋白质表达水平明显增高(P<0.01)。结论化学合成的siRNA在短时期内可有效地抑制TIMP-1基因的表达,重组病毒rAAV/siRNA-TIMP-1/neo可长期有效地抑制TIMP-1基因表达。

Objective To construct recombinant adeno-associated virus （AAV） carrying small interfering RNA （siRNA） of tissue inhibitor of metalloproteinase-1 （T IMP-1 ） and investigate the long-term effect of TIMP-1 gene RNA interference on rat hepatic stellate cell（HSC）-T6 cells and the influence on the expression of matrix metalloproteinase（MMP） 13 in vitro. Methods U6 promotor followed by the 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/neo. After infection of this recombinant AAV into rat HSC-T6 cells and selection by G418, real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene and MMP13 gene in HSC-T6 cells at 4 weeks and 12 weeks. Results The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pd16-95/siRNA-TIMP-1/neo had been l^econstructed successfully. After it had been packed in 293 cell to form rAAV/siRNA-TIMP-1/neo and infected into HSC-T6, the transcription and expression level of TIMP-1 in HSC-T6 cells which were infected by the rAAV/siRNA-TIMP-1/neo were suppressed dramatically compared with mock control and normal HSC-T6 cells （ P 〈 0.01 ）, and transcription and expression level of MMP13 in HSC-T6 cells infected by the rAAV/siRNA-TIMP-1/neo were increased significantly compared with mock control and normal HSC-T6 cells （ P 〈 0.01 ）. Conclusion RNA interference can cause suppression of TIMP-1 gene in rat HSC, and when this function combined with AAV infection it can suppress the specific gene expression for long time.