调节TREK1对小鼠海马神经干细胞增殖和BDNF表达的影响
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摘要
目的:观察调节TWIK相关的K+通道1(TREK1)对小鼠海马神经干细胞增殖和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)表达的影响。方法:从孕10.5 d C57bl/6J小鼠胚胎海马中分离出神经干细胞并培养,待细胞达到70%～80%融合后,对细胞进行慢病毒干预,细胞分为sham组,Ctrl组(转染对照病毒),Plenti-TREK-1组(转染携带TREK1高表达载体病毒)和sh-TREK-1组(转染携带TREK1-shRNA病毒)。采用CCK-8法检测病毒干预后3天和7天各组神经干细胞的细胞活力,采用q RT-PCR检测病毒干预后7天各组神经干细胞TREK-1基因表达情况,并通过Elisa检测各组神经干细胞上清液BDNF表达水平。结果:与sham组相比较,(1)Plenti-TREK-1组TREK1基因表达上调,神经干细胞的神经球体积减小,细胞活力降低,细胞增殖减少,细胞上清BDNF水平下降;(2)sh-TREK-1组TREK1基因表达下调,神经干细胞的神经球体积增加,细胞活力增高,细胞增殖增加,细胞上清BDNF水平上调;(3)上述各项指标Ctrl组与sham组之间无统计学差异。结论:神经干细胞的增殖与TREK1通道密切相关,上调TREK-1可以抑制神经干细胞增殖及其BDNF的表达水平;下调TREK-1则可以促进神经干细胞增殖并上调其BDNF的表达水平。
Objective: To investigate the effects of TREK-1 over-express or knock-down on the proliferation and brain derived neurotrophic factor(BDNF) expression in neural stem cells from the mice hippocampus. Methods: Neural stem cells were isolated from the embryonic hippocampus of 10.5 D C57 bl/6 J mice and cultured. After the cells reached 70% ～ 80% fusion, the cells were subjected to lentiviral intervention and divided into sham group, Ctrl group, Plenti-TREK-1 group and sh-TREK-1 group. The cell viability in each group was detected by CCK-8 method at 3 and 7 days after virus intervention. The expression of TREK-1 gene and BDNF in the supernatant was detected by QRT-PCR or Elisa at 7 days after virus intervention respectively. Results:(1) Compared with sham group, the m RNA level of TREK1 was up-regulated and the volume of neurospheres, cell activity and cell proliferation as well as the level of BDNF in the supernatant were reduced in Plenti-TREK-1 group;(2) the m RNA level of TREK1 was down-regulated and the volume of neurospheres, cell activity and cell proliferation as well as the level of BDNF in the supernatant were increased in sh-TREK-1 group when compared with sham group;(3) There were no significant differences between sham and Ctrl group in the above parameters. Conclusion:The proliferation of neural stem cells is mediated by the expression of TREK1 channel. Up-regulation of TREK-1 inhibits the proliferation of neural stem cells and the expression of BDNF, and down-regulation of TREK-1 promotes the proliferation of neural stem cells and up-regulate the expression of BDNF.
Objective: To investigate the effects of TREK-1 over-express or knock-down on the proliferation and brain derived neurotrophic factor(BDNF) expression in neural stem cells from the mice hippocampus. Methods: Neural stem cells were isolated from the embryonic hippocampus of 10.5 D C57 bl/6 J mice and cultured. After the cells reached 70% ～ 80% fusion, the cells were subjected to lentiviral intervention and divided into sham group, Ctrl group, Plenti-TREK-1 group and sh-TREK-1 group. The cell viability in each group was detected by CCK-8 method at 3 and 7 days after virus intervention. The expression of TREK-1 gene and BDNF in the supernatant was detected by QRT-PCR or Elisa at 7 days after virus intervention respectively. Results:(1) Compared with sham group, the m RNA level of TREK1 was up-regulated and the volume of neurospheres, cell activity and cell proliferation as well as the level of BDNF in the supernatant were reduced in Plenti-TREK-1 group;(2) the m RNA level of TREK1 was down-regulated and the volume of neurospheres, cell activity and cell proliferation as well as the level of BDNF in the supernatant were increased in sh-TREK-1 group when compared with sham group;(3) There were no significant differences between sham and Ctrl group in the above parameters. Conclusion:The proliferation of neural stem cells is mediated by the expression of TREK1 channel. Up-regulation of TREK-1 inhibits the proliferation of neural stem cells and the expression of BDNF, and down-regulation of TREK-1 promotes the proliferation of neural stem cells and up-regulate the expression of BDNF.
引文
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[23]Cai Y,Peng Z,Guo H,et al.TREK-1 pathway mediates isofluraneinduced memory impairment in middle-aged mice[J].Neurobiology of learning and memory,2017,145:199-204
[24]Mazella J,Petrault O,Lucas G,et al.Spadin,a sortilin-derived peptide,targeting rodent TREK-1 channels:a new concept in the antidepressant drug design[J].PLo S biology,2010,8(4):e1000355
[25]韦朝霞.BDNF-Trk B通路在海马脑片培养中的神经再生研究[D]南方医科大学,2013
[26]Zuccato C,Liber D,Ramos C,et al.Progressive loss of BDNF in a mouse model of Huntington's disease and rescue by BDNF delivery[J].Pharmacological research,2005,52(2):133-139
[27]孟盼,王宇红,张秀丽,等.百事乐胶囊对慢性应激抑郁模型大鼠行为活动及海马突触素表达的影响[J].世界科学技术:中医药现代化,2013,15(7):1562-1568
[2]Baptista P,Andrade J P.Adult Hippocampal Neurogenesis:Regulation and Possible Functional and Clinical Correlates[J].Frontiers in neuroanatomy,2018,12:44
[3]Akers K G,Cherasse Y,Fujita Y,et al.Concise Review:Regulatory Influence of Sleep and Epigenetics on Adult Hippocampal Neurogenesis and Cognitive and Emotional Function[J].Stem cells,2018[Epub ahead of print]
[4]Rotheneichner P,Lange S,O'sullivan A,et al.Hippocampal neurogenesis and antidepressive therapy:shocking relations[J].Neural plasticity,2014,2014:723915
[5]Toda T,Parylak S L,Linker S B,et al.The role of adult hippocampal neurogenesis in brain health and disease[J].Molecular psychiatry,2018[Epub ahead of print]
[6]Blurton-Jones M,Kitazawa M,Martinez-Coria H,et al.Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(32):13594-13599
[7]Ehling P,Cerina M,Budde T,et al.The CNS under pathophysiologic attack——examining the role of K(2)p channels[J].Pflugers Archiv:European journal of physiology,2015,467(5):959-972
[8]Zhang M,Yin H J,Wang W P,et al.Over-expressed human TREK-1inhibits CHO cell proliferation via inhibiting PKA and p38 MAPKpathways and subsequently inducing G1 arrest[J].Acta pharmacologica Sinica,2016,37(9):1190-1198
[9]Xi G,Zhang X,Zhang L,et al.Fluoxetine attenuates the inhibitory effect of glucocorticoid hormones on neurogenesis in vitro via a two-pore domain potassium channel,TREK-1[J].Psychopharmacology,2011,214(3):747-759
[10]Ye D,Li Y,Zhang X,et al.TREK1 channel blockade induces an antidepressant-like response synergizing with 5-HT1A receptor signaling[J].European neuropsychopharmacology:the journal of the European College of Neuropsychopharmacology,2015,25(12):2426-2436
[11]Zhou C H,Zhang Y H,Xue F,et al.Isoflurane exposure regulates the cell viability and BDNF expression of astrocytes via upregulation of TREK1[J].Molecular medicine reports,2017,16(5):7305-7314
[12]Kempermann G.Activity Dependency and Aging in the Regulation of Adult Neurogenesis[J].Cold Spring Harbor perspectives in biology,2015,7(11):a018929
[13]Urban N,Guillemot F.Neurogenesis in the embryonic and adult brain:same regulators,different roles[J].Frontiers in cellular neuroscience,2014,8:396
[14]Christian K M,Song H,Ming G L.Functions and dysfunctions of adult hippocampal neurogenesis[J].Annual review of neuroscience,2014,37:243-262
[15]Lim D A,Alvarez-Buylla A.The Adult Ventricular-Subventricular Zone(V-SVZ)and Olfactory Bulb(OB)Neurogenesis[J].Cold Spring Harbor perspectives in biology,2016,8(5):a018820
[16]Kaneko N,Sawada M,Sawamoto K.Mechanisms of neuronal migration in the adult brain[J].Journal of neurochemistry,2017,141(6):835-847
[17]Schoenfeld T J,Gould E.Stress,stress hormones,and adult neurogenesis[J].Exp Neurol,2012,233(1):12-21
[18]Vieira M S,Santos A K,Vasconcellos R,et al.Neural stem cell differentiation into mature neurons:Mechanisms of regulation and biotechnological applications[J].Biotechnology advances,2018,36(7):1946-1970
[19]Maljevic S,Lerche H.Potassium channels:a review of broadening therapeutic possibilities for neurological diseases[J].Journal of neurology,2013,260(9):2201-2211
[20]Mao Q,Yuan J,Ming X,et al.Role of dorsal root ganglion K2p1.1 in peripheral nerve injury-induced neuropathic pain[J].Molecular pain,2017,13:1744806917701135
[21]Noel J,Sandoz G,Lesage F.Molecular regulations governing TREKand TRAAK channel functions[J].Channels,2011,5(5):402-409
[22]Olsen M L,Khakh B S,Skatchkov S N,et al.New Insights on Astrocyte Ion Channels:Critical for Homeostasis and Neuron-Glia Signaling[J].The Journal of neuroscience:the official journal of the Society for Neuroscience,2015,35(41):13827-13835
[23]Cai Y,Peng Z,Guo H,et al.TREK-1 pathway mediates isofluraneinduced memory impairment in middle-aged mice[J].Neurobiology of learning and memory,2017,145:199-204
[24]Mazella J,Petrault O,Lucas G,et al.Spadin,a sortilin-derived peptide,targeting rodent TREK-1 channels:a new concept in the antidepressant drug design[J].PLo S biology,2010,8(4):e1000355
[25]韦朝霞.BDNF-Trk B通路在海马脑片培养中的神经再生研究[D]南方医科大学,2013
[26]Zuccato C,Liber D,Ramos C,et al.Progressive loss of BDNF in a mouse model of Huntington's disease and rescue by BDNF delivery[J].Pharmacological research,2005,52(2):133-139
[27]孟盼,王宇红,张秀丽,等.百事乐胶囊对慢性应激抑郁模型大鼠行为活动及海马突触素表达的影响[J].世界科学技术:中医药现代化,2013,15(7):1562-1568