NR1基因RNA干扰的海马神经干细胞体系的构建

The construction of hippocampal neural stem cell system interfered by NR1 RNAi gene

目的:构建NR1基因RNA干扰(RNA interference,RNAi)的海马神经干细胞体系,用于离体水平研究NR1对精神分裂症模型海马神经发生的调控作用。方法:利用293T细胞,将病毒原液稀释至不同病毒梯度,经过逐孔稀释法检测慢病毒滴度;体外培养海马神经干细胞,分别加入MOI值为100、10、1的慢病毒,确定海马神经干细胞的最适MOI值;海马神经干细胞体系中加入浓度为200μmol/L的MK-801 24 h后,加入干扰NR1亚基的慢病毒12 h,通过荧光显微镜观察海马神经干细胞干扰效率,通过Western Blot检测转染后海马神经干细胞NR1的蛋白表达。结果:(1)重组的慢病毒滴度可达2×10~8~2×10~9TU/m L。MOI值为100时,海马神经干细胞转染率可达90%;(2)Western Blot结果显示:感染慢病毒的海马神经干细胞中NR1蛋白表达水平明显降低(P<0.05)。结论:成功构建了NR1基因RNA干扰的海马神经干细胞体系,慢病毒介导的shRNA可有效的干扰海马神经干细胞中NR1亚基的表达,为进一步探讨NR1对精神分裂症海马神经发生调控机制的研究提供了依据。

Objective： To build NR1 gene RNA interference （RNA interference, RNAi） system of hippocampal neural stem cells in vitro, which was used to study the regulation of NR1 on hippocampal neurogenesis in schizophrenia models. Methods： Taking use of 293T cells to dilute the virus into different viral gradients which were tested by the method of porous dilution. In vitro the cultured hippocampal neural stem cells were added the lentivirus at the MOI of 100, 10 and 1 respectively to found the optimal MOI value of the hippocampal stem cells. Twenty four hours after adding 200μmol/L MK-801 into the media of hippocampal neural stem cell, lentivirus carrying the interference NR1 subunit were added into the same media for 12 hours. Then, the transfection efficiency of hippocampal neural stem cell were observed by fluorescence microscope, the NR1 protein expression of hippocampal neural stem cells was detected by Western Blot. Results ： （ 1 ） The drop of reorganized lentivirus could be 2× 10s - 2 × 109 TU/ml. When MOI value is 100, the transfection rate of hippoeampal stem cell was up to 90% ; （2）The Western Blot results showed that the expression level of NR1 protein in the hippocampal stem cells infected by the lentivirus decreased significantly （ P 〈0. 05 ）. Conclusions： The NR1 gene RNA interference system was established successfully. The lentivirus carrying shRNA of NR1 can effectively interfere the NR1 subunit expression in hippocampal neural stem cells, which provide the basis for further investigation of the regulation mechanism of NR1 on hippoeampal neurogenesis in schizophrenia.