光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响
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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DCXChR2-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Neu N+阳性细胞(成熟神经元标志物)的密度和Neu N+/Hoechst比值情况; Western blot检测各组成熟神经元相关蛋白MAP2、Neu N、Neurog2、Neuro D1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Western blot检测各组MAP2、Neu N、Neurog2、Neuro D1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Neu N+阳性细胞密度(成熟细胞)和Neu N+/Hoechst明显高于NSCs组和NSCs+EGFP组(P均<0.05); Western blot检测的MAP2、Neu N、Neurog2、Neuro D1、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、Neuro D1和GluR2蛋白表达水平明显下降(P均<0.01),Neu N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 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.
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 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.
引文
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[15]Abdanipour A,Noori-Zadeh A,Mesbah-Namin SA,et al.Di-(2-ethylhexyl)phthalate-induced hippocampus-derived neural stem cells proliferation[J].Cell J,2017,19(1):166-172.
[16]Taylor NE,Van Dort CJ,Kenny JD,et al.Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia[J].Proc Natl Acad Sci U S A,2016,113(45):12826-12831.
[17]Packer AM,Roska B,Hausser M.Targeting neurons and photons for optogenetics[J].Nat Neurosci,2013,16(7):805-815.
[18]Cheng X,Li Y,Huang Y,et al.Pulse labeling and longterm tracing of newborn neurons in the adult subgranular zone[J].Cell Res,2011,21(2):338-349.
[19]王凌星,黄红红,吕国荣.产前850-1900MHz手机暴露对子代大鼠齿状回PCNA和DCX表达的影响[J].中国应用生理学杂志,2018,31(4):35-42.
[20]Ming GL,Song H.Adult neurogenesis in the mammalian brain:significant answers and significant questions[J].Neuron,2011,70(4):687-702.
[21]Walker TL,Yasuda T,Adams DJ,et al.The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronallineage cells[J].J Neurosci,2007,27(14):3734-3742.
[22]Karl C,Couillard-Despres S,Prang P,et al.Neuronal precursor-specific activity of a human doublecortin regulatory sequence[J].J Neurochem,2005,92(2):264-282.
[23]颜春鲁,李盛华,安方玉,等.右归丸对鼠膝骨性关节炎的治疗作用及Wnt信号通路相关因子表达的影响[J].中国应用生理学杂志,2018,34(2):177-181.
[24]Kondo T,Matsuoka AJ,Shimomura A,et al.Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3[J].Stem Cells,2011,29(5):836-846.
[25]Hodge RD,Hevner RF.Expression and actions of transcription factors in adult hippocampal neurogenesis[J].Dev Neurobiol,2011,71(8):680-689.
[2]Stocchetti N.Traumatic brain injury:problems and opportunities[J].Lancet Neurol,2014,13(1):14-16.
[3]Sahay A,Scobie KN,Hill AS,et al.Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation[J].Nature,2011,472(7344):466-470.
[4]Song J,Zhong C,Bonaguidi MA,et al.Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision[J].Nature,2012,489(7414):150-154.
[5]Chen C,Ma TZ,Wang LN,et al.Mild hypothermia facilitates the long-term survival of newborn cells in the dentate gyrus after traumatic brain injury by diminishing a pro-apoptotic microenvironment[J].Neuroscience,2016,335:114-121.
[6]Zhao WY,Chen SB,Wang JJ,et al.Establishment of an ideal time window model in hypothermic-targeted temperature management after traumatic brain injury in rats[J].Brain Res,2017,1669:141-149.
[7]Deisseroth K,Singla S,Toda H,et al.Excitation-neurogenesis coupling in adult neural stem/progenitor cells[J].Neuron,2004,42(4):535-552.
[8]Bost A,Shaib AH,Schwarz Y,et al.Large dense-core vesicle exocytosis from mouse dorsal root ganglion neurons is regulated by neuropeptide Y[J].Neuroscience,2017,346:1-13.
[9]Lee HC,Lim S,Han JY.Wnt/beta-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro[J].Sci Rep,2016,6:34510.
[10]Spitzer NC.Electrical activity in early neuronal development[J].Nature,2006,444(7120):707-712.
[11]D'Ascenzo M,Piacentini R,Casalbore P,et al.Role of L-type Ca2+channels in neural stem/progenitor cell differentiation[J].Eur J Neurosci,2006,23(4):935-944.
[12]牛江涛,张泽国,曹瑞,等.四逆散对PTSD及睡眠障碍大鼠海马CA1/CA3区神经元动作电位的影响[J].中国应用生理学杂志,2018,34(4):363-366.
[13]Emery B.Regulation of oligodendrocyte differentiation and myelination[J].Science,2010,330(6005):779-782.
[14]Spangler SM,Bruchas MR.Optogenetic approaches for dissecting neuromodulation and GPCR signaling in neural circuits[J].Curr Opin Pharmacol,2017,32:56-70.
[15]Abdanipour A,Noori-Zadeh A,Mesbah-Namin SA,et al.Di-(2-ethylhexyl)phthalate-induced hippocampus-derived neural stem cells proliferation[J].Cell J,2017,19(1):166-172.
[16]Taylor NE,Van Dort CJ,Kenny JD,et al.Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia[J].Proc Natl Acad Sci U S A,2016,113(45):12826-12831.
[17]Packer AM,Roska B,Hausser M.Targeting neurons and photons for optogenetics[J].Nat Neurosci,2013,16(7):805-815.
[18]Cheng X,Li Y,Huang Y,et al.Pulse labeling and longterm tracing of newborn neurons in the adult subgranular zone[J].Cell Res,2011,21(2):338-349.
[19]王凌星,黄红红,吕国荣.产前850-1900MHz手机暴露对子代大鼠齿状回PCNA和DCX表达的影响[J].中国应用生理学杂志,2018,31(4):35-42.
[20]Ming GL,Song H.Adult neurogenesis in the mammalian brain:significant answers and significant questions[J].Neuron,2011,70(4):687-702.
[21]Walker TL,Yasuda T,Adams DJ,et al.The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronallineage cells[J].J Neurosci,2007,27(14):3734-3742.
[22]Karl C,Couillard-Despres S,Prang P,et al.Neuronal precursor-specific activity of a human doublecortin regulatory sequence[J].J Neurochem,2005,92(2):264-282.
[23]颜春鲁,李盛华,安方玉,等.右归丸对鼠膝骨性关节炎的治疗作用及Wnt信号通路相关因子表达的影响[J].中国应用生理学杂志,2018,34(2):177-181.
[24]Kondo T,Matsuoka AJ,Shimomura A,et al.Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3[J].Stem Cells,2011,29(5):836-846.
[25]Hodge RD,Hevner RF.Expression and actions of transcription factors in adult hippocampal neurogenesis[J].Dev Neurobiol,2011,71(8):680-689.
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