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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,so
2、astoestablishasetofreliabletechniquefortheisolationandpurificationoftheastrocytescerebralcortex.METHODS:TheexperimentwasperformedintheInstituteofLifeScience,TaishanMedicalCollegefromJunetoAugust2006.Wistarratsaged2-3daysafterbirthofeithersexwereprovidedbyExperimentalAnimalCen
3、ter,InstituteofLifeScience,TaishanMedicalCollege,andselectedtocarryoutprimarycultureofcerebralastrocytes.Theyweredividedintotwogroups:routineculturegroupanddifferentialvelocityadherentgroup.Astrocyteswereremovedatminutes15and30inthelattergroup,andthencultureflaskwasrotatedtor
4、emovesupernatantintoanothercultureflask,andthenkeptinincubatorforfollowingculture.After7-10days,eachgroupwaspassagedtillcellsgrewintodemixing,andthenputinswingbedat37℃andshookat250r/minfor18hours.Supernatantwasadded,andthenthecellswerewashedwithD-Hank'sliquorthreetimes,andtre
5、atedwith0.25%trypsinization,observedunderinvertedmicroscope.Mediumcontainingserumwasaddedtostopdigestionwhenprocessofcellsrecovered.Thecellswereblownandhitbypipetteforkeepingcellsfromflaskwall,andthencentrifugedat1000r/minfor5minutes.SupernatantwasremovedbeforeDMEMmediumconta
6、ining0.2volumefractionwassuspendedandprecipitated.ThecellswereincubatedinL-polylysinecultureflasksuccessively.Doubleimmunofluorescencestainingwasselectedtoidentifypurificationoftheastrocytes.Atthesametime,integratedopticaldensity(IOD)wasusedtomeasuregrowthstateofastrocytes.RE
7、SULTS:①Purityofastrocytescouldbeelevatedmarkedlybydifferentialvelocityadherenttechnique[routineculturegroup:(82±3)%,differentialvelocityadherentgroupof15minutes:(94±2)%,differentialvelocityadherentgroupof30minutes:(95±2)%,P<0.01.Differentialvelocityadherenttechniqueneededenou
8、ghtime.Therewasnosignificantdifferenceinelevatingcellpurityinthe15mi
