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1、周欣,明曉云,康頌建,白波,段耀奎.差速貼壁技術對大鼠腦皮質星形膠質細胞純化率的影響.中國組織工程研究與臨床康復,2007,11(15):2829-2831.InfluenceofdifferentialvelocityadherenttechniqueonthepurifiedrateofcerebralastrocytesinratsAbstractAIM:Toobservetheproportionofratcerebralastrocyteculturedbydifferentialvelocityadherenttechnique,soastoestablishaseto
2、freliabletechniquefortheisolationandpurificationoftheastrocytescerebralcortex.METHODS:TheexperimentwasperformedintheInstituteofLifeScience,TaishanMedicalCollegefromJunetoAugust2006.Wistarratsaged2-3daysafterbirthofeithersexwereprovidedbyExperimentalAnimalCenter,InstituteofLifeScience,TaishanMe
3、dicalCollege,andselectedtocarryoutprimarycultureofcerebralastrocytes.Theyweredividedintotwogroups:routineculturegroupanddifferentialvelocityadherentgroup.Astrocyteswereremovedatminutes15and30inthelattergroup,andthencultureflaskwasrotatedtoremovesupernatantintoanothercultureflask,andthenkeptini
4、ncubatorforfollowingculture.After7-10days,eachgroupwaspassagedtillcellsgrewintodemixing,andthenputinswingbedat37℃andshookat250r/minfor18hours.Supernatantwasadded,andthenthecellswerewashedwithD-Hank'sliquorthreetimes,andtreatedwith0.25%trypsinization,observedunderinvertedmicroscope.Mediumcontai
5、ningserumwasaddedtostopdigestionwhenprocessofcellsrecovered.Thecellswereblownandhitbypipetteforkeepingcellsfromflaskwall,andthencentrifugedat1000r/minfor5minutes.SupernatantwasremovedbeforeDMEMmediumcontaining0.2volumefractionwassuspendedandprecipitated.ThecellswereincubatedinL-polylysinecultu
6、reflasksuccessively.Doubleimmunofluorescencestainingwasselectedtoidentifypurificationoftheastrocytes.Atthesametime,integratedopticaldensity(IOD)wasusedtomeasuregrowthstateofastrocytes.RESULTS:①Purityofastrocytescouldbeelevatedmarkedlybydifferentialvelocityadherenttechnique[routineculturegroup:
7、(82±3)%,differentialvelocityadherentgroupof15minutes:(94±2)%,differentialvelocityadherentgroupof30minutes:(95±2)%,P<0.01.Differentialvelocityadherenttechniqueneededenoughtime.Therewasnosignificantdifferenceinelevatingcellpurityinthe15mi
