Lingo-1介导Rho-ROCK通路调控神经干细胞的分化
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摘要
背景:Lingo-1在神经元轴突再生、髓鞘形成等过程中具有重要作用,但其是否参与对神经干细胞分化的调控以及具体机制尚无定论。目的:观察干预神经干细胞Lingo-1表达水平对其分化方向的影响,并探索其可能机制。方法:从新生大鼠脑组织分离培养神经干细胞,经神经干细胞标志物检测鉴定后分别使用Lingo-1 shRNA慢病毒载体及阴性对照病毒转染神经干细胞,再用成神经元诱导分化培养基培养7 d,随后运用免疫荧光、免疫RhoA印迹技术检测神经元相关标志物表达,并检测、ROCK1蛋白表达水平。结果与结论:(1)从新生大鼠脑组织所分离培养的神经干细胞呈浮球状生长,高表达神经干细胞特异性标志物Nestin、Sox2与Pax6,符合神经干细胞特征;(2)慢病毒载体转染可在mRNA及蛋白质水平实现对神经干细胞Lingo-1的干扰,经诱导培养7 d后Lingo-1敲低的神经干细胞分化为神经元的比例显著增加,且伴随着RhoA、ROCK1蛋白表达水平的下降;(3)结果表明,Lingo-1参与对神经干细胞分化方向的调控,抑制Lingo-1可促进其向神经元方向分化,该过程可能与调控Rho-ROCK通路有关。
BACKGROUND: It has been reported that Lingo-1 plays an important role in neuronal axon regeneration and myelination, but whether it participates in the regulation of neural stem cell differentiation remains unknown. OBJECTIVE: To examine the effect of Lingo-1 knockdown on the differentiation of neural stem cells and to explore its potential mechanism.METHODS: Neural stem cells were isolated from neonatal rat brain tissue and identified by determination of several neural stem cell specific markers. Then the cells were transfected with Lingo-1 sh RNA lentivirus or negative control vectors, and were further cultured in induced differentiation medium for 7 days. Immunofluorescence and western blot techniques were thereafter used to detect the expression of neuron specific markers and to examine expression levels of Rho A and ROCK1. RESULTS AND CONCLUSION: The cells isolated from brain tissue of neonatal rats aggregated to form non-adherent spherical clusters with high expression of Nestin, Sox2, and Pax6, specific markers of neural stem cells, which exhibited the characteristics of neural stem cells. After transfected with lentivirus, expression of Lingo-1 was successfully knocked down at m RNA and protein levels. After 7 days of induction, expression of neuron specific markers was significantly increased in Lingo-1 knocked-down neural stem cells. Meanwhile, expressions of Rho A and ROCK1 were decreased. Thus, Lingo-1 can be involved in the differentiation of neural stem cells. Inhibition of Lingo-1 can induce neural stem cells to differentiate into neurons, which is possibly related to the regulation of Rho-ROCK signaling pathway.
BACKGROUND: It has been reported that Lingo-1 plays an important role in neuronal axon regeneration and myelination, but whether it participates in the regulation of neural stem cell differentiation remains unknown. OBJECTIVE: To examine the effect of Lingo-1 knockdown on the differentiation of neural stem cells and to explore its potential mechanism.METHODS: Neural stem cells were isolated from neonatal rat brain tissue and identified by determination of several neural stem cell specific markers. Then the cells were transfected with Lingo-1 sh RNA lentivirus or negative control vectors, and were further cultured in induced differentiation medium for 7 days. Immunofluorescence and western blot techniques were thereafter used to detect the expression of neuron specific markers and to examine expression levels of Rho A and ROCK1. RESULTS AND CONCLUSION: The cells isolated from brain tissue of neonatal rats aggregated to form non-adherent spherical clusters with high expression of Nestin, Sox2, and Pax6, specific markers of neural stem cells, which exhibited the characteristics of neural stem cells. After transfected with lentivirus, expression of Lingo-1 was successfully knocked down at m RNA and protein levels. After 7 days of induction, expression of neuron specific markers was significantly increased in Lingo-1 knocked-down neural stem cells. Meanwhile, expressions of Rho A and ROCK1 were decreased. Thus, Lingo-1 can be involved in the differentiation of neural stem cells. Inhibition of Lingo-1 can induce neural stem cells to differentiate into neurons, which is possibly related to the regulation of Rho-ROCK signaling pathway.
引文
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[23]Wu X,Walker CL,Lu Q,et al.Rho A/Rho Kinase Mediates Neuronal Death Through Regulating c PLA2 Activation.Mol Neurobiol.2017;54(9):6885-6895.
[24]辛延乐,李艳花,张辉,等.Rho激酶抑制剂:治疗神经系统变性疾病的潜在药物[J].中国神经免疫学和神经病学杂志,2015,22(4):288-290.
[25]Ding J,Li QY,Wang X,et al.Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice.J Neurochem.2010;114(6):1619-1629.
[26]Liu C,Li Y,Yu J,et al.Targeting the shift from M1 to M2macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil.PLo S One.2013;8(2):e54841.
[27]Hou SW,Liu CY,Li YH,et al.Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis,acting possibly through antiinflammatory effect.CNSNeurosci Ther.2012;18(11):909-917.
[28]Chen N,Cen JS,Wang J,et al.Targeted Inhibition of Leucine-Rich Repeat and Immunoglobulin Domain-Containing Protein 1 in Transplanted Neural Stem Cells Promotes Neuronal Differentiation and Functional Recovery in Rats Subjected to Spinal Cord Injury.Crit Care Med.2016;44(3):e146-157.
[29]Andrews JL,Fernandez-Enright F.A decade from discovery to therapy:Lingo-1,the dark horse in neurological and psychiatric disorders.Neurosci Biobehav Rev.2015;56:97-114.
[30]Zhang Y,Zhang YP,Pepinsky B,et al.Inhibition of LINGO-1promotes functional recovery after experimental spinal cord demyelination.Exp Neurol.2015;266:68-73.
[2]Bond AM,Ming GL,Song H.Adult Mammalian Neural Stem Cells and Neurogenesis:Five Decades Later.Cell Stem Cell.2015;17(4):385-395.
[3]Hong JY,Lee SH,Lee SC,et al.Therapeutic potential of induced neural stem cells for spinal cord injury.J Biol Chem.2014;289(47):32512-32525.
[4]G?ritz C,Frisén J.Neural stem cells and neurogenesis in the adult.Cell Stem Cell.2012;10(6):657-659.
[5]Yoneyama T,Arai MA,Akamine R,et al.Notch Inhibitors from Calotropis gigantea That Induce Neuronal Differentiation of Neural Stem Cells.J Nat Prod.2017;80(9):2453-2461.
[6]Zhang X,He X,Li Q,et al.PI3K/AKT/m TOR Signaling Mediates Valproic Acid-Induced Neuronal Differentiation of Neural Stem Cells through Epigenetic Modifications.Stem Cell Reports.2017;8(5):1256-1269.
[7]Faigle R,Song H.Signaling mechanisms regulating adult neural stem cells and neurogenesis.Biochim Biophys Acta.2013;1830(2):2435-2448.
[8]索磊,杨印祥,汤文燕,等.Lingo-1在中枢神经系统损伤疾病中的应用[J].中华神经医学杂志,2016,15(4):421-424.
[9]Okafuji T,Tanaka H.Expression pattern of LINGO-1 in the developing nervous system of the chick embryo.Gene Expr Patterns.2005;6(1):57-62.
[10]马英,陆莹.Lingo-1研究进展及其对神经系统疾病的相关影响[J].中国神经免疫学和神经病学杂志,2016,23(4):287-292.
[11]Yin W,Hu B.Knockdown of Lingo1b protein promotes myelination and oligodendrocyte differentiation in zebrafish.Exp Neurol.2014;251:72-83.
[12]L??v C,Fernqvist M,Walmsley A,et al.Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons.PLo S One.2012;7(1):e29771.
[13]Connor B.Concise Review:The Use of Stem Cells for Understanding and Treating Huntington's Disease.Stem Cells.2018;36(2):146-160.
[14]Reis C,Wilkinson M,Reis H,et al.A Look into Stem Cell Therapy:Exploring the Options for Treatment of Ischemic Stroke.Stem Cells Int.2017;2017:3267352.
[15]Xiong Z,Zhao S,Mao X,et al.Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation.Stem Cell Res.2014;12(2):387-399.
[16]Hou S,Lu P.Direct reprogramming of somatic cells into neural stem cells or neurons for neurological disorders.Neural Regen Res.2016;11(1):28-31.
[17]Shao Z,Lee X,Huang G,et al.LINGO-1 Regulates Oligodendrocyte Differentiation through the Cytoplasmic Gelsolin Signaling Pathway.J Neurosci.2017;37(12):3127-3137.
[18]Gresle MM,Liu Y,Kilpatrick TJ,et al.Blocking LINGO-1 in vivo reduces degeneration and enhances regeneration of the optic nerve.Mult Scler J Exp Transl Clin.2016;2:2055217316641704.
[19]Sun JJ,Ren QG,Xu L,et al.LINGO-1 antibody ameliorates myelin impairment and spatial memory deficits in experimental autoimmune encephalomyelitis mice.Sci Rep.2015;5:14235.
[20]Wang J,Ye Z,Zheng S,et al.Lingo-1 sh RNA and Notch signaling inhibitor DAPT promote differentiation of neural stem/progenitor cells into neurons.Brain Res.2016;1634:34-44.
[21]Mi S,Miller RH,Lee X,et al.LINGO-1 negatively regulates myelination by oligodendrocytes.Nat Neurosci.2005;8(6):745-751.
[22]韩佳寅,易艳,梁爱华,等.Rho/ROCK信号通路研究进展[J].药学学报,2016,51(6):853-859.
[23]Wu X,Walker CL,Lu Q,et al.Rho A/Rho Kinase Mediates Neuronal Death Through Regulating c PLA2 Activation.Mol Neurobiol.2017;54(9):6885-6895.
[24]辛延乐,李艳花,张辉,等.Rho激酶抑制剂:治疗神经系统变性疾病的潜在药物[J].中国神经免疫学和神经病学杂志,2015,22(4):288-290.
[25]Ding J,Li QY,Wang X,et al.Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice.J Neurochem.2010;114(6):1619-1629.
[26]Liu C,Li Y,Yu J,et al.Targeting the shift from M1 to M2macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil.PLo S One.2013;8(2):e54841.
[27]Hou SW,Liu CY,Li YH,et al.Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis,acting possibly through antiinflammatory effect.CNSNeurosci Ther.2012;18(11):909-917.
[28]Chen N,Cen JS,Wang J,et al.Targeted Inhibition of Leucine-Rich Repeat and Immunoglobulin Domain-Containing Protein 1 in Transplanted Neural Stem Cells Promotes Neuronal Differentiation and Functional Recovery in Rats Subjected to Spinal Cord Injury.Crit Care Med.2016;44(3):e146-157.
[29]Andrews JL,Fernandez-Enright F.A decade from discovery to therapy:Lingo-1,the dark horse in neurological and psychiatric disorders.Neurosci Biobehav Rev.2015;56:97-114.
[30]Zhang Y,Zhang YP,Pepinsky B,et al.Inhibition of LINGO-1promotes functional recovery after experimental spinal cord demyelination.Exp Neurol.2015;266:68-73.