一种改进的大鼠皮层神经元原代培养方法及其性质鉴定_姜茜.pdf

( ) ·212 · JOURNALOFPEKINGUNIVERSITY(HEALTHSCIENCES)　Vo.l4 1　No.2　Ap.r2009 ·技术方法 · 一种改进的大鼠皮层神经元原代培养方法 及其性质鉴定 ■ 姜　茜, 姜玉武 , 王静敏, 秦　炯, 吴希如 (, 　1000 34 ) [　] 目的:, 、、 。方法: 3, 16 ～ 17 d Wistar, , 。 4 ～6 h B27 Neurobasal , 3(DIV3) 10 μ ol/L (cytosinearabinosid,eAra-C) 24 h , 1。 , 2 ( icrotubule-associatedprotein2, MAP2 ), 、。结果:, , , , , , , 、, 。结论:, , 。 [] ;;, ;; [] R329.2　　[] A　　[] 1671-167X(2009 )02-02 12-05 do:i10.3969/.jissn.167 1-167 X.2009.02.019 Aniproved ethodforpriarycultureofratcorticalneuronandcellidentification ■ JIANGQian, JIANGYu-wu, WANGJing- in, QINJiong, WUXi-ru (DepartentofPediatric,sPekingUniversityFirstHospita,lBeijing100034, China) ABSTRACT　Objectiv:eToiproveprevious ethodofpriaryratcorticalneuronculturetogetpurer and orelong-lastingcellsforstudy.Methods:Tied-pregnantWistarratsatagestationalageof16 or 17 days(16 -17 d)wereused.Fetalbrainswerereovedandthecerebralcorticesweredissectedou.t Papaindigestionand echanicaldissociationwerecobinedtoconductono-cellsuspending edia. Fourtosixhours(4-6 h)post-plating, allplating ediawerereovedfro culturesandreplacedwith Neurobasalediu suppleentedwithB27.Onthethirdday, 10 μ ol/Lcytosinearabinoside(Ara-C) wasaddedtotheculturefor24 htoinhibittheoutgrowthofglialcell.sHalfoftheculture ediuwas changedeveryweek.The orphologicalchangesofneuroncellswereobservedbylight icroscope. Doublei uno-stainingoficrotubule-associatedprotein2 (MAP2 )andkaryonwereappliedtoassess theculturepurity.Evaluationofsynapseforationwasprocessedbyi unocytocheicalanalysisusing antibodiesagainstbothpre-andpostsynapticprotein arker.sResult:sTheiproved ethodcouldre- arkablyincreasethecellnuberandreduceneuronaldanification.Thepriaryculturewascharacte- rizedbyhighunifority, purity, noralsynapseforation