靶向大鼠海马神经干细胞STAT3慢病毒shRNA干扰载体的构建与鉴定

Construction and identification of shRNA lentiviral vector targeting STAT3 in rat hippocampus NSCs

目的:构建与鉴定靶向大鼠海马干细胞(neural stem cells,NSCs)信号转导与转录激活因子3(signal transducer and activator of transcription factor3,STAT3)shRNA慢病毒载体。方法:设计并合成4条靶向大鼠STAT3基因的shRNA干扰序列(shRNA1、2、3、4)及1条阴性对照序列(negative control,NC),将其分别重组于慢病毒载体,慢病毒包装后感染大鼠海马神经干细胞,荧光显微镜下通过观察绿色荧光蛋白(green fluorescent protein,GFP)表达计算慢病毒感染效率;利用RT-PCR和Western Blot检测干扰后大鼠海马NSCs中STAT3的mRNA和蛋白质表达水平。结果:干扰序列测序结果正确;感染指数(multiply of index,MOI)为1∶20时感染效率最高为85.6±3.14%;感染大鼠海马NSCs 72 h后利用RT-PCR和Western Blot检测干扰效率发现shRNA1干扰效率最好,其基因水平干扰效率为81.3%±4.22%;蛋白水平干扰效率为72.3%±5.12%。结论:成功构建靶向大鼠海马干细胞STAT3慢病毒干扰载体,并通过筛选发现shRNA1能有效抑制NSCs的STAT3 mRNA和蛋白表达,为研究海马NSCs分化机制奠定了工作基础。

Objective: Construct and identify signal transducer and activator of transcription factor 3(STAT3) shRNA lentiviral vector targeting STAT3 in rat hippocampus neural stem cells(NSCs). Method: Four pairs of rat STAT3 shRNA oligoes(shRNA1,shRNA 2,shRNA 3,shRNA 4 and negative control) were designed and synthesized. Oligoes were recombined into the lentiviral vector,and the lentivirus which were packaged infected rat hippocampus NSCs. Lentivirus infection efficiency was calculated by green fluorescent protein(GFP) expression under fluorescence microscope; RT-PCR and Western Blot were used to detect mRNA and protein level of STAT3 in rat hippocampus NSCs after being interfered.Results: The accuracy of sequenced shRNA lentiviral vector were verified,and the infection efficiency of shRNA lentiviral vector was 85. 6 ± 3. 14% when multiply of index(MOI) was 1∶ 20. The results of RT-PCR and Western Blot showed that the shRNA1 has the best interference efficiency targeting on STAT3 in rat hippocampus NSCs,which downregulated STAT3 mRNA by 81. 3 ± 4. 22% and lowered STAT3 protein level by 72. 3 ± 5. 12% in NSCs by 72 h after transfection.Conclusion: We successfully constructing the shRNA lentiviral vector targeting on STAT3 expression in rat hippocampus NSCs; We observe that shRNA1 recombinant lentiviral vector could suppress the expression of STAT3 mRNA and proteinin NSCs. The present study provides a solide foundation for the subsequent study of hippocampal NSCs differentiation mechanism.