Xia Tian-Guang, Zhu Xu, Wang Jing-Jing, Wei Meng-Guang, Lyu Fang-Fang, Chen Chong, Liang Jun, Jiang Wei, Sun Qian, Sun Hong-Tao
Institute of Traumatic Brain Injury and Neurology, Logistics University of Chinese People Armed Police Forces, Tianjin 300162.
Department of Neurosurgery, Handan Central Hospital, Hebei 056002.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2019 May 28;35(3):256-261. doi: 10.12047/j.cjap.5731.2019.054.
To investigate the effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway.
Neural stem cells (ESCs)were extracted from the cerebral cortex of fetal rat and transfected by lentivirus carrying DCX-ChR2-EGFP gene and the expression of DCX of newborn neurons differentiated from neural stem cells were observed. All cells were divided into 3 groups(n=9): control group, NSCs+EGFP and NSCs+ChR2 groups. The control group was normal cultured NSCs (NSCs group); the neural stem cells in NSCs+EGFP group were transfected with lentivirus carrying EGFP gene. The neural stem cells in NSCs+ChR2 group were infected with lentivirus carrying DCX-ChR2-EGFP gene. After 48 hours of lentivirus infection, 470 nm blue laser irradiation was performed for 3 consecutive days. NeuN positive cell density(the maturation of neural stem cells)and the ratio of NeuN/Hoechst in each group were observed. Western blot was used to detect the expression levels of MAP2, NeuN, Neurog2, NeuroD1 and GluR2. Western blot was used to detect the expressions of β-catenin and TCF4 associated with Wnt/β-catenin signaling channel. Verapamil (100 μmol/L, L-type calcium channel blockers) and Dkk1 (50 μg/ml, β-catenin inhibitor) were used to treat stem cells of the NSCs+ChR2 group and then the expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were detected by Western blot.
After 3 days of 470 nm blue laser irradiation, NeuN positive cell density(the maturation of neural stem cells)and the ratio of NeuN/Hoechst, the expression levels of the protein MAP2, NeuN, Neurog2, NeuroD1, GluR and the protein β-catenin and TCF4 associated with Wnt/β-catenin signaling channel detected by Western blot were significantly increased in the group of NSCs+ChR2, compared with NSCs and NSCs+EGFP groups. The expressions of MAP2, NeuN, Neurog2, NeuroD1 and GluR were remarkably decreased after treated by verapamil and Dkk1 in the group of NSCs+ChR2. It was proved that the opening of ChR2 channel producing cationic influx promoted the maturation of neural stem cells and induced by the Wnt/β-catenin signaling pathway.
Optical genetic promoted the maturation of newborn neurons through the Wnt/β-catenin signaling pathway.
通过Wnt/β-连环蛋白通路研究光遗传学技术对新生神经元的影响。
从胎鼠大脑皮层提取神经干细胞（ESCs），用携带DCX-ChR2-EGFP基因的慢病毒转染，观察神经干细胞分化的新生神经元中DCX的表达。所有细胞分为3组（n=9）：对照组、NSCs+EGFP组和NSCs+ChR2组。对照组为正常培养的神经干细胞（NSCs组）；NSCs+EGFP组的神经干细胞用携带EGFP基因的慢病毒转染。NSCs+ChR2组的神经干细胞用携带DCX-ChR2-EGFP基因的慢病毒感染。慢病毒感染48小时后，连续3天进行470nm蓝光照射。观察每组NeuN阳性细胞密度（神经干细胞成熟度）及NeuN/Hoechst比值。采用蛋白质免疫印迹法检测MAP2、NeuN、Neurog2、NeuroD1和GluR2的表达水平。采用蛋白质免疫印迹法检测与Wnt/β-连环蛋白信号通路相关的β-连环蛋白和TCF4的表达。用维拉帕米（100μmol/L，L型钙通道阻滞剂）和Dkk1（50μg/ml，β-连环蛋白抑制剂）处理NSCs+ChR2组干细胞，然后用蛋白质免疫印迹法检测MAP2、NeuN、Neurog2、NeuroD1和GluR的表达。
470nm蓝光照射3天后，NSCs+ChR2组NeuN阳性细胞密度（神经干细胞成熟度）及NeuN/Hoechst比值、蛋白质免疫印迹法检测的蛋白质MAP2、NeuN、Neurog2、NeuroD1、GluR以及与Wnt/β-连环蛋白信号通路相关的蛋白质β-连环蛋白和TCF4的表达水平均显著高于NSCs组和NSCs+EGFP组。NSCs+ChR2组经维拉帕米和Dkk1处理后，MAP2、NeuN、Neurog2、NeuroD1和GluR的表达明显降低。证明ChR2通道开放产生阳离子内流促进神经干细胞成熟，且由Wnt/β-连环蛋白信号通路介导。
光遗传学通过Wnt/β-连环蛋白信号通路促进新生神经元成熟。
