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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,soas
2、toestablishasetofreliabletechniquefortheisolationandpurificationoftheastrocytescerebralcortex.METHODS:TheexperimentwasperformedintheInstituteofLifeScience,TaishanMedicalCollegefromJunetoAugust2006.Wistarratsaged2-3daysafterbirthofeithersexwereprovidedbyExperimentalAnimalCenter,
3、InstituteofLifeScience,TaishanMedicalCollege,andselectedtocarryoutprimarycultureofcerebralastrocytes.Theyweredividedintotwogroups:routineculturegroupanddifferentialvelocityadherentgroup.Astrocyteswereremovedatminutes15and30inthelattergroup,andthencultureflaskwasrotatedtoremoves
4、upernatantintoanothercultureflask,andthenkeptinincubatorforfollowingculture.After7-10days,eachgroupwaspassagedtillcellsgrewintodemixing,andthenputinswingbedat37℃andshookat250r/minfor18hours.Supernatantwasadded,andthenthecellswerewashedwithD-Hank'sliquorthreetimes,andtreatedwith
5、0.25%trypsinization,observedunderinvertedmicroscope.Mediumcontainingserumwasaddedtostopdigestionwhenprocessofcellsrecovered.Thecellswereblownandhitbypipetteforkeepingcellsfromflaskwall,andthencentrifugedat1000r/minfor5minutes.SupernatantwasremovedbeforeDMEMmediumcontaining0.2vo
6、lumefractionwassuspendedandprecipitated.ThecellswereincubatedinL-polylysinecultureflasksuccessively.Doubleimmunofluorescencestainingwasselectedtoidentifypurificationoftheastrocytes.Atthesametime,integratedopticaldensity(IOD)wasusedtomeasuregrowthstateofastrocytes.RESULTS:①Purit
7、yofastrocytescouldbeelevatedmarkedlybydifferentialvelocityadherenttechnique[routineculturegroup:(82±3)%,differentialvelocityadherentgroupof15minutes:(94±2)%,differentialvelocityadherentgroupof30minutes:(95±2)%,P<0.01.Differentialvelocityadherenttechniqueneededenoughtime.Therewa
8、snosignificantdifferenceinelevatingcellpurityinthe15mi
