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1、周欣,明晓云,康颂建,白波,段耀奎.差速贴壁技术对大鼠脑皮质星形胶质细胞纯化率的影响.中国组织工程研究与临床康复,2007,11(15):2829-2831.InfluenceofdifferentialvelocityadherenttechniqueonthepurifiedrateofcerebralastrocytesinratsAbstractAIM:Toobservetheproportionofratcerebralastrocyteculturedbydifferentialvelocityadherenttechnique,soastoesta
2、blishasetofreliabletechniquefortheisolationandpurificationoftheastrocytescerebralcortex.METHODS:TheexperimentwasperformedintheInstituteofLifeScience,TaishanMedicalCollegefromJunetoAugust2006.Wistarratsaged2-3daysafterbirthofeithersexwereprovidedbyExperimentalAnimalCenter,InstituteofL
3、ifeScience,TaishanMedicalCollege,andselectedtocarryoutprimarycultureofcerebralastrocytes.Theyweredividedintotwogroups:routineculturegroupanddifferentialvelocityadherentgroup.Astrocyteswereremovedatminutes15and30inthelattergroup,andthencultureflaskwasrotatedtoremovesupernatantintoanot
4、hercultureflask,andthenkeptinincubatorforfollowingculture.After7-10days,eachgroupwaspassagedtillcellsgrewintodemixing,andthenputinswingbedat37℃andshookat250r/minfor18hours.Supernatantwasadded,andthenthecellswerewashedwithD-Hank'sliquorthreetimes,andtreatedwith0.25%trypsinization,obse
5、rvedunderinvertedmicroscope.Mediumcontainingserumwasaddedtostopdigestionwhenprocessofcellsrecovered.Thecellswereblownandhitbypipetteforkeepingcellsfromflaskwall,andthencentrifugedat1000r/minfor5minutes.SupernatantwasremovedbeforeDMEMmediumcontaining0.2volumefractionwassuspendedandpre
6、cipitated.ThecellswereincubatedinL-polylysinecultureflasksuccessively.Doubleimmunofluorescencestainingwasselectedtoidentifypurificationoftheastrocytes.Atthesametime,integratedopticaldensity(IOD)wasusedtomeasuregrowthstateofastrocytes.RESULTS:①Purityofastrocytescouldbeelevatedmarkedly
7、bydifferentialvelocityadherenttechnique[routineculturegroup:(82±3)%,differentialvelocityadherentgroupof15minutes:(94±2)%,differentialvelocityadherentgroupof30minutes:(95±2)%,P<0.01.Differentialvelocityadherenttechniqueneededenoughtime.Therewasnosignificantdifferenceinelevatingcellpur
8、ityinthe15mi
