特异性shRNA对小鼠海马神经干细胞中Olig2基因表达的影响

Effects of specific sh RNA on the expression of Olig2 gene in mouse hippocampal neural stem cells

目的:构建RNA干扰(RNAi)少突胶质细胞转录因子2(Olig2)基因的小鼠海马神经干细胞(NSCs)体系,用于离体水平研究Olig2对精神分裂症模型海马神经发生的调控作用。方法:海马NSCs原代培养及鉴定;通过荧光显微镜分别观察24、48、72 h后NSCs感染情况,确定慢病毒感染的最佳时间;通过Western Blot筛选有效干扰Olig2的shRNA序列;在NSCs体系中加入浓度为200μmol/L的MK-801 24 h后,加入有效干扰Olig2的shRNA,通过Western Blot检测慢病毒感染后的Olig2蛋白表达。结果:慢病毒感染72 h后,海马神经干细胞球荧光表达最强,可达90%;Western Blot结果显示:与对照组比较,Olig2 shRNA3干扰组Olig2的蛋白表达明显降低(P <0. 05);干扰组可明显降低MK-801处理后的NSCs中Olig2蛋白表达(P <0. 05)。结论:成功构建RNA干扰Olig2基因的海马NSCs体系,为进一步研究Olig2对精神分裂症后海马神经发生的调控机制奠定了基础。

Objective: To construct oligodenrocyte transcription factor 2(Olig2) gene RNA interference(RNAi) system of hippocampal neural stem cells in vitro and further to study the regulation mechanism of Olig2 on hippocampal neurogenesis in schizophrenia. Methods:Primary culture and identification of hippocampal neural stem cells;At the different time points of 24,48,72 h after adding lentivirus into the media of hippocampal neural stem cells to found the optimal infection time by fluorescence microscope;After adding different lentivirus into the media of hippocampal neural stem cells to found the optimal lentivirus sequence by Western Blot;Twenty four hours after adding200 μmol/L MK-801 into the media of hippocampal neural stem cell,lentivirus carrying the interference Olig2 subunit were added into the same media,the Olig2 protein expression of hippocampal neural stem cells was detected by Western Blot. Results: Immunofluorescence showed that the cell fluorescence was the strongest at 72 h after infection and the transfection rate of hippocampal stem cell was to 90%;Compared with the control,the expression of Olig2 protein decreased significantly in the Olig2 shRNA3 group(P < 0. 05);the expression level of Olig2 protein in the hippocampal stem cells infected by the lentivirus decreased significantly(P < 0. 05). Conclusion: The Olig2 gene RNAi system was established successfully. The lentivirus carrying shRNA of Olig2 can effectively interference the Olig2 protein expression in hippocampal neural stem cells,which provide the basis for further investigation of the regulation mechanism of Olig2 on hippocampal neurogenesis in schizophrenia.