| 345 | 0 | 39 |
| 下载次数 | 被引频次 | 阅读次数 |
本研究通过体外分离培养及鉴定原代小鼠小脑颗粒神经元(cerebellar granule neurons,CGNs),为神经科学研究提供原代神经元细胞模型。取生后第7天的ICR乳鼠小脑,在解剖体视镜下剥离脑膜及血管,经刀片切碎、0.25%胰酶消化、移液器吹打、70μm尼龙滤网制备单细胞悬液,差速贴壁后接种于新鲜配置的培养基。倒置相差显微镜观察不同时间CGNs的形态变化。采用神经元的标记蛋白微管相关蛋白-2(microtubule associated protein 2,MAP2),星形胶质细胞的标记蛋白胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP),小胶质细胞的标记蛋白(type 3 complement receptor,CR3/CD11b)进行免疫荧光检测培养的原代CGNs纯度。原代培养CGNs接种20 min后即贴壁良好,培养24 h后细胞伸出突起,培养第7天神经元成熟,形成丰富的轴突,树突和胞间突触连接。经免疫荧光鉴定,CGNs纯度可达95%以上。成功体外分离培养出高纯度的原代CGNs,可应用于CGNs的体外研究。
Abstract：Isolation and identification of primary culture of cerebellar granule neurons(CGNs) in vitro, for neuroscience experiments to provide primary neurons model. Cerebellum was removed from a postnatal 7 days old ICR mouse, meninges and blood vessels were cleaned carefully under stereoscopic microscope. The cerebella were minced with surgical blade, digested with 0.25% trypsin, dissociated by pipetting and filtered with 70 μm nylon mesh to obtain single cell suspension. Granule cells were purified by differential velocity adherence method and cultured in fresh medium. The morphological change of cultures at differential stages was observed under phasecontrast microscope. Characterization and purity of the neurons were determined by immunostaining of neuron marker(microtubule associated protein 2, MAP2), astrocyte marker(glial fibrillary acidic protein, GFAP) and microglia marker(type 3 complement receptor, CR3/CD11b). Isolated CGNs adhered well to the substrate after 20 mins,cells started to extend axons and dendrites after 24 hours. The neurons matured and form neural network at day 7.The purity of cultured CGNs was determined to be more than 95 percent by using immunofluorescent technique.The high purity primary culture of CGNs had been successfully obtained and could be used for in vitro studies.
D'ange lo E.,2013,Ce rebellar g ranule cell,In:Manto M.,Schmahmann J.D.,Rossi F.,Gruol D.L.,and Koibuchi N.(eds.),Handbook of the cerebellum and cerebellar disorders,Springer,Netherlands,pp.765-791
Giordano G.,and Costa L.G.,2011,Primary neurons in culture and neuronal cell lines for in vitro neurotoxicological studies,Methods Mol.Biol.,758:13-27
Guijarro P.,Jiang J.,and Yuan X.B.,2012,Culturing of cerebellar granule cells to study neuronal migration:gradient and local perfusion assays,Curr.Protoc.Neurosci.,Chapter 3(3),Unit 3.26:3.26.1-3.26.12
Kouzoukas D.E.,Li G.Y.,Takapoo M.,Moninger T.,Bhalla R.C.and Pantazis N.J.,2013,Intracellular calcium plays a critical role in the alcohol-mediated death of cerebellar granule neurons,J.Neurochem.,124(3):323-335
Kramer D.,and Minichiello L.,2010,Cell culture of primary cerebellar granule cells,Methods Mol.Biol.,633:233-239
Manto M.,Bower J.M.,Conforto A.B.,Delgado-Garcia J.M.,Da Guarda S.N.F.,Gerwig M.,Habas C.,Hagura N.,Ivry R.B.,Marien P.,Molinari M.,Naito E.,Nowak D.A.,Oulad Ben Taib N.,Pelisson D.,Tesche C.D.,Tilikete C.,and Timmann D.,2012,Consensus paper:roles of the cerebellum in motor control--the diversity of ideas on cerebellar involvement in movement,Cerebellum,11(2):457-487
Millen K.J.,and Gleeson J.G.,2008,Cerebellar development and disease,Curr.Opin.Neurobiol.,18(1):12-19
Selvakumar T.,and Kilpatrick D.L.,2013,Culturing mouse cerebellar granule neurons,Methods Mol.Biol.,1018:49-59
Steinmetz A.B.,and Free man J.H.,2016,Cannabinoid modulation of memory consolidation within the cerebellum,Neurobiol.Learn Mem.,136:228-235
Tapiero H.,MathéG.,Couvreur P.,and Tew K.D.,2002,ⅡGlutamine and glutamate,Biomed,Pharmacother.,56(9):446-457
Wang L.,and Zhu Z.A.,2014,Nitric oxide show its survival role by NO-PKC pathway through c GMP-dependent or independent on the culture of cerebella granular neurons,Neurosci.Lett.,583:165-169
Yao H.J.,Zhou L.W.,Pei J.R.,Liu X.N.,and Wang J.,2013,Primary culture of rat cerebellar granule neurons in vitro,Zhonghua Difangbingxue Zazhi(Chinese Journal of Endemiology),32(1):38-41(姚洪菊,周令望,裴俊瑞,刘晓娜,王静,2013,大鼠小脑颗粒神经元体外原代培养,中华地方病学杂志,32(1):38-41)
Kim Y.H.,Baek N.S.,Han Y.H.,Chung M.A.and Jung S.D.2011,Enhancement of neuronal cell adhesion by covalen binding of poly-d-lysine,J.Neurosci.Methods,202(1):38-44
Yu A.C.H.,Chan P.H.,and Fishman R.A.,1987,Arachidonic acid inhibits uptake of glutamate and glutamine but not of GABA in cultured cerebellar granule cells,J.Neurosci.Res.,17(4):424-427
Yu A.C.H.,Drejer J.,Hertz L.,and Schousboe A.,1983,Pyruvate carboxylase activity in primary cultures of astrocytes and neurons,J.Neurochem.,41(5):1484-1487
基本信息:
DOI：10.13417/j.gab.037.002615
中图分类号:R338
引用信息:
[1]罗道朋,许寒晓,李强明,等.小鼠小脑颗粒神经元的原代培养与鉴定[J].基因组学与应用生物学,2018,37(06):2615-2620.DOI:10.13417/j.gab.037.002615.
基金信息:
贵州省科技厅社会发展攻关项目(黔科合SY字(2014)3024号);; 贵阳市科技计划项目(筑科合同(20141001)21号);; 贵州省科学技术基金(黔科合J字(2015)2012号);; 贵州省科技合作计划项目(黔科合LH字(2015)7348号)基金共同资助
2018-06-25
2018-06-25