光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响*

doi:10.12047/j.cjap.5731.2019.054

中国应用生理学杂志 ›› 2019, Vol. 35 ›› Issue (3): 256-261.doi: 10.12047/j.cjap.5731.2019.054

光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响^*

夏天光¹, 朱旭^{1, 2}, 王景景¹, 魏孟广¹, 吕方方¹, 陈翀¹, 梁军³, 姜伟³, 孙倩³, 孙洪涛^1△

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

出版日期:2019-05-28 发布日期:2019-06-28
通讯作者: Tel: 13702167676; E-mail: chenmo333@163.com
基金资助:
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)

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

XIA Tian-guang¹, ZHU Xu^1,2, WANG Jing-jing¹, WEI Meng-guang¹, LYU Fang-fang¹, CHEN Chong¹, LIANG Jun³, JIANG Wei³, SUN Qian³, SUN Hong-Tao^1△

1. Institute of Traumatic Brain Injury and Neurology, Logistics University of Chinese People Armed Police Forces, Tianjin 300162;
2. Department of Neurosurgery, Handan Central Hospital, Hebei 056002;
3. Institute of Medical Engineering and Transformation Medicine, Tianjin University, Tianjin 300072, China

Online:2019-05-28 Published:2019-06-28

摘要/Abstract

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48 h后连续3 d行470 nm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;Western blot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100 μmol/L维拉帕米或50 μg/ml的β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞,然后行Western blot检测各组MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平。结果:连续3 d 470 nm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);Western blot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。

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

Abstract: 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 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. Results: 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. Conclusion: Optical genetic promoted the maturation of newborn neurons through the Wnt/β-catenin signaling pathway.

Key words: optical genetic, Wnt/β-catenin signaling pathway, ChR2 channel protein, DCX, newborn neurons

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. 光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响^*[J]. 中国应用生理学杂志, 2019, 35(3): 256-261.

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. The effects of optical genetic techniques on new neurons through the Wnt/β-Catenin pathway[J]. CJAP, 2019, 35(3): 256-261.

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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

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