调节TREK1对小鼠海马神经干细胞增殖和BDNF表达的影响

目的:观察调节TWIK相关的K+通道1(TREK1)对小鼠海马神经干细胞增殖和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)表达的影响。方法:从孕10.5 d C57bl/6J小鼠胚胎海马中分离出神经干细胞并培养,待细胞达到70%～80%融合后,对细胞进行慢病毒干预,细胞分为sham组,Ctrl组(转染对照病毒),Plenti-TREK-1组(转染携带TREK1高表达载体病毒)和sh-TREK-1组(转染携带TREK1-shRNA病毒)。采用CCK-8法检测病毒干预后3天和7天各组神经干细胞的细胞活力,采用q RT-PCR检测病毒干预后7天各组神经干细胞TREK-1基因表达情况,并通过Elisa检测各组神经干细胞上清液BDNF表达水平。结果:与sham组相比较,(1)Plenti-TREK-1组TREK1基因表达上调,神经干细胞的神经球体积减小,细胞活力降低,细胞增殖减少,细胞上清BDNF水平下降;(2)sh-TREK-1组TREK1基因表达下调,神经干细胞的神经球体积增加,细胞活力增高,细胞增殖增加,细胞上清BDNF水平上调;(3)上述各项指标Ctrl组与sham组之间无统计学差异。结论:神经干细胞的增殖与TREK1通道密切相关,上调TREK-1可以抑制神经干细胞增殖及其BDNF的表达水平;下调TREK-1则可以促进神经干细胞增殖并上调其BDNF的表达水平。

Regulation of TREK1 on the Proliferation and BDNF Expression in the Hippocampal Neural Stem Cells of Mice

ABSTRACT Objective: To investigate the effects of TREK-1 over-express or knock-down on the proliferation and brain derived neurotrophic factor (BDNF) expression in neural stem cells from the mice hippocampus. Methods: Neural stem cells were isolated from the embryonic hippocampus of 10.5 D C57bl/6J mice and cultured. After the cells reached 70% ~ 80% fusion, the cells were subjected to lentiviral intervention and divided into sham group, Ctrl group, Plenti-TREK-1 group and sh-TREK-1 group. The cell viability in each group was detected by CCK-8 method at 3 and 7 days after virus intervention. The expression of TREK-1 gene and BDNF in the super- natant was detected by QRT-PCR or Elisa at 7 days after virus intervention respectively. Results: (1) Compared with sham group, the mRNA level of TREK1 was up-regulated and the volume of neurospheres, cell activity and cell proliferation as well as the level of BDNF in the supernatant were reduced in Plenti-TREK-1 group; (2) the mRNA level of TREK1 was down-regulated and the volume of neuro- spheres, cell activity and cell proliferation as well as the level of BDNF in the supernatant were increased in sh-TREK-1 group when compared with sham group; (3) There were no significant differences between sham and Ctrl group in the above parameters. Conclusion: The proliferation of neural stem cells is mediated by the expression of TREK1 channel. Up-regulation of TREK-1 inhibits the prolifera- tion of neural stem cells and the expression of BDNF, and down-regulation of TREK-1 promotes the proliferation of neural stem cells and up-regulate the expression of BDNF.