光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响  

The effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway

作　　者：夏天光 朱旭 王景景 魏孟广 吕方方 陈翀 梁军 姜伟 孙倩 孙洪涛 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;Institute of Medical Engineering and Transformation Medicine,Tianjin University,Tianjin 300072,China)

机构地区：[1]武警特色医学中心脑创伤与神经疾病研究所,天津300162 [2]邯郸市中心医院神经外科,河北056002 [3]天津大学医学工程与转化医学研究院,天津300072

出　　处：《中国应用生理学杂志》2019年第3期256-261,I0001,共7页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金(81771352,81671222,81771350);天津市救援医学临床医学研究中心课题(15ZXLCSY 00040)

摘　　要：目的:探讨Wnt/β-catenin信号通路光遗传技术在促进新生神经元成熟中的作用。方法:从胎鼠大脑皮层中提取神经干细胞,用携带DCX-ChR2-EGFP基因的慢病毒感染神经干细胞,观察神经干细胞分化为新生神经元后DCX的表达。实验细胞分为3组(n=9):对照组、NSCs+EGFP和NSCs+ChR2组。其中对照组为正常培养的NSCs(NSCs组);NSCs+EGFP组为携带DCX-EGFP基因慢病毒感染神经干细胞组;NSCs+ChR2组为携带DCX-ChR2-EGFP基因慢病毒感染神经干细胞组。病毒感染后48h后连续3d行470nm蓝激光照射,然后检测各组NeuN+阳性细胞(成熟神经元标志物)的密度和NeuN+/Hoechst比值情况;Westernblot检测各组成熟神经元相关蛋白MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平和Wnt/β-catenin通道相关蛋白TCF4和β-catenin蛋白的表达水平。用L-型钙通道阻断剂100μmol/L维拉帕米或50μg/ml的β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞,然后行Westernblot检测各组MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平。结果:连续3d470nm蓝激光照射后,NSCs+ChR2组中NeuN+阳性细胞密度(成熟细胞)和NeuN+/Hoechst明显高于NSCs组和NSCs+EGFP组(P均<0.05);Westernblot检测的MAP2、NeuN、Neurog2、NeuroD1、GluR2蛋白及Wnt/β-catenin通路相关蛋白β-catenin、TCF4表达水平均明显高于NSCs组和NSCs+EGFP组(P均<0.01);L-型钙通道阻断剂维拉帕米或β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞后MAP2、Neurog2、NeuroD1和GluR2蛋白表达水平明显下降(P均<0.01),NeuN表达水平也下降(P<0.05)。证明ChR2通道蛋白开放产生阳离子内流促进新生神经元成熟,是通过Wnt/β-catenin信号通路实现的。结论:光遗传学方法通过Wnt/β-catenin信号通路促进新生神经元成熟。Objective: To investigate the effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway. Methods: Neural stem cells( ESCs) were extracted from the cerebral cortex of fetal rat and transfected by lentivirus carrying DCXChR2-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. Neu N+positive cell density( the maturation of neural stem cells) and the ratio of Neu N+/Hoechst in each group were observed. Western blot was used to detect the expression levels of MAP2,Neu N,Neurog2,Neuro D1 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,"-catenin inhibitor) were used to treat stem cells of the NSCs+ChR2 group and then the expressions of MAP2,Neu N,Neurog2,Neuro D1 and GluR were detected by Western blot. Results:After 3 days of 470 nm blue laser irradiation,Neu N+positive cell density( the maturation of neural stem cells) and the ratio of Neu N+/Hoechst,the expression levels of the protein MAP2,Neu N,Neurog2,Neuro D1,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,Neu N,Neurog2,Neuro D1 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

关 键 词：光遗传学 WNT/Β-CATENIN信号通路 ChR2通道蛋白 DCX 新生神经元 

分 类 号：R3[医药卫生—基础医学]