肾母细胞瘤过度表达基因对大鼠肝星状细胞MMP-1和TIMP-1表达的作用研究

Effects of nephroblastoma overexpressed gene on expression of MMP-1 and TIMP-1 of rat hepatic stellate cells

目的构建能表达靶向肾母细胞瘤过度表达基因(NOV)的小干扰RNA(siRNA)的重组质粒,研究NOV对大鼠肝星状细胞(HSC)增殖、基质金属蛋白酶-1(MMP-1)和金属蛋白酶组织抑制因子-1(TIMP-1)表达的影响。方法以NOV为目的基因,以质粒psiRNA-hH1neo为载体,构建能在真核细胞中表达的靶向NOV的siRNA的重组质粒psiRNA(psiRNA1、2、3)和阴性对照重组质粒pconsiRNA。限制性酶切和测序鉴定构建成功后,脂质体介导重组质粒转染HSC,依据转染质粒的不同将HSC分为psiRNA1组、psiRNA2组、psiRNA3组、阴性对照pconsiRNA组,并以未转染重组质粒的HSC为空白对照组。半定量RT-PCR检测HSC的NOV、MMP-1、TIMP-1 mRNA表达情况,MTT法检测细胞增殖。结果限制性酶切和测序鉴定表明成功构建了NOV siRNA表达质粒;与阴性对照组相比,转染外源重组质粒psiRNA2的HSC内NOV的mRNA表达水平下降(P<0.05),MMP-1 mRNA表达水平下降不明显(P>0.05),而TIMP-1 mRNA表达水平明显下降(P<0.05)。与空白对照组相比,转染外源重组质粒psiRNA2的HSC增殖活性显著降低(P<0.05)。psiRNA1组、psiRNA3组的HSC内NOV、MMP-1和TIMP-1 mRNA的表达及HSC增殖活性均无明显下降(P均>0.05)。结论NOV可促进HSC增殖及表达TIMP-1增加,而对MMP-1表达影响不大,提示NOV可以作为肝纤维化基因治疗的一个新的靶位点。

Objective To construct recombinant plasmids that can express small interfering RNA （siRNA） targeting nephroblastoma overexpressed gene （NOV） and to investigate the effects of NOV on the biological behaviors of hepatic stellate cells （HSCs）. Methods Hairpin siRNA templates targeting NOV gene were synthesized and cloned into plasmid vector psiRNA-Hlneo. Three vector-derived siRNAs （denoted psiRNA1, 2 and 3） and a mocking pconsiRNA （as control） were constructed. The recombinant NOV siRNA plasmids were constructed and identified by using restrictive enzyme analysis and DNA sequencing, and were then transfected into HSCs by lipofectamine. HSCs were divided into group psiRNA1, psiRNA2, psiRNA3 and pconsiRNA and were transfected with recombinant plasmids. Control group consisted of HSCs containing no plasmids. Expression of NOV, matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 were detected by semi-quantitative RT-PCR. Cell proliferation was assayed by MTT method. Results Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Compared with control group, extrogenous recombinant plasmid psiRNA2 could reduce the level of NOV mRNA （P 〈 0.05） and tissue inhibitor of metalloproteinase-1 mRNA （P 〈 0.05 ） , yet failed to decrease the expression of matrix metalloproteinase-1 mRNA （P 〉 0.05）. Compared with non-transfection group, extrogenous recombinant plasmid psiRNA2 significantly decreased the proliferative activity of HSC （P 〈 0.05 ）. No obvious changes were found in psiRNA1 group and psiRNA3 group （P 〉0.05 in both）. Conclusion NOV can increase the secretion of tissue inhibitor of metalloproteinase-1 and promote the proliferation of HSC. NOV may be a novel target for gene therapy of liver fibrosis.