大鼠TIMP1 miRNA慢病毒RNAi载体的构建及其对HSC-T6细胞TIMP1表达影响的研究

Construction of lentiviral vector of the rnai mirna of RAT-TIMP1-GENE and whose Impact on the TIMP1 gene expression in HSC-T6 cells

目的构建大鼠TIMP1 miRNA慢病毒RNAi载体,并观察其转染HSC-T6细胞后对TIMP1基因表达的影响。方法根据BLOCK-iT Poll miR RNAi Expression System with EmGFP要求,应用在线miRNA设计工具http://rnaidesigner.invitrogen.com/rnaiexpress/,针对大鼠TIMP1基因(GenBank:U06179)的598位点设计、合成Oligo DNA,退火形成双链DNA后,与载体pcDNATM6.2-GW/EmGFP-miR连接,转化Top10感受态细胞,构建TIMP1 miRNA慢病毒RNAi载体。经PCR扩增及测序鉴定正确后,以脂质体LipofectimineTM2000介导转染经TGF-β1刺激的大鼠肝星状细胞株HSC-T6,24、48、72h后收集细胞,应用RT-PCR鉴定TIMP1的表达情况。结果经PCR及测序鉴定构建的TIMP1 miRNA慢病毒RNAi载体正确;将其转染经TGF-β1刺激的大鼠肝星状细胞株HSC-T6,48 h即可见TIMP1 mRNA表达量下降,72 h检测不到TIMP1 mRNA的表达,而未转染组及转染pLacZ-miR/GFP(阴性对照)组未见此改变。结论成功构建大鼠TIMP1 miR-NA慢病毒RNAi载体pTIMP1-miR/GFP;pTIMP1-miR/GFP使大鼠肝星状细胞株HSC-T6 TIMP1基因沉默。提示了RNA干扰可使肝纤维化形成过程中的关键因子TIMP1基因沉默,为RNA干扰技术进一步应用治疗大鼠肝纤维化模型奠定了基础。

Objective To construct lentiviral vector of the RNAi miRNA of rat-TIMP1- gene, and observe the TIMP1 gene expression in HSC-T6 cells that is transfected by the vector. Methods Oligo DNA designed by online nfiRNA Design Tool（http：// maidesigner, invitrogen, com/maiexpress/） which is designed to silence 598 site of rat-TIMP1-gene（GenBank： U06179） and to meet the requirement of BLOCK- iT Poll miR RNAi Expression System with EmGFP, is transformed into competent cell after synthesis, renaturattion to form double strand DNA and Connection with the wDNA 6.2-GW/EmGFP-miR vector to construct lentiviral vector of the RNAi miRNA of rat-TIMP1-gene, the vector is transformed into rat hepatic stellate cells（HSC-T6） stimulanted by TGF-β1 mediated by the lipidosome Lipofectimine 2000 after whose sequence proved correct after PCR amplification and sequencing, then we collect the cells after 24 h,48 h and 72 h, and identify the TIMP1 expression by RT-PCR. Results Sequence of lentiviral vector of the RNAi miRNA of rat-TIMP1-gene is proved correct by PCR amplification and sequencing; After the vector being transfected into Rat hepatic stellate cells（HSC-T6） stimulanted by TGF-β1, we observed TIMP mRNA expressing decline at 48 h, and no TIMP mRNA expression was detected at 72 h,but we did not observed the same change from the rat hepatic stellate cells（HSC-T6） without vector transfected and Rat hepatic stellate cells（HSC-T6） with pLacZ-miR/GFP transfected（negative control）. Conclusion Lentiviral vector of the RNAi nfiRNA of rat-TIMP1-gene pTIMPI-nfiR/GFP was constructed successfully; pTIMP1-miR/GFP（lentiviral vectorof the RNAi miRNA of rat-TIMP1-gene） silence the TIMP1 gene of the Rat hepatic stellate cells（HSC-T6）. Our results demonstrated that RNA interference is able to silence the key gene TIMP1 in the process of the formation of hepatic fibrosis, thus we could explore healing hepatic fibrosis of rat via RNA interference teetmology.