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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 investigate the expression of TIMP-1 after HSC-T6 infection with adeno-associated virus (AAV) as a vector containing small interfering RNA (siRNA) targeting the tissue inhibitor of metalloproteinase 1 (TIMP-1) Matrix metalloproteinase 13 (MMP13) expression. METHODS: A pair of siRNAs with the strongest inhibitory effect on rat TIMP-1 gene was constructed into short hairpin siRNA expression vector in vitro and then packaged into recombinant AAV-rAAV / siRNA-TIMP-1 / neo and infected with HSC -T6. The mRNA and protein expression of TIMP-1 and MMP13 were detected by fluorescence quantitative PCR and Western blot at 4 weeks and 12 weeks after infection. Results The most potent siRNA against shRNA was confirmed by PCR, restriction enzyme digestion and sequencing. The shRNA expression vector was successfully cloned in vitro. Compared with the control group, the mRNA and protein expression of TIMP-1 in rAAV / siRNA-TIMP-1 / neo group were significantly decreased at 4 and 12 weeks after infection (P <0.05) P <0.01), while MMP13 mRNA and protein expression levels were significantly increased (P <0.01). Conclusion The chemically synthesized siRNA can effectively inhibit the expression of TIMP-1 gene in a short period of time. Recombinant virus rAAV / siRNA-TIMP-1 / neo can inhibit TIMP-1 gene expression for a long time.