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1、中国组织工程研究与临床康复第13卷第6期2009–02–05出版JournalofClinicalRehabilitativeTissueEngineeringResearchFebruary5,2009Vol.13,No.6BasicMedicineIsolationofrabbitbonemarrowmesenchymalstemcellsusingdensitygradientcentrifugationandadherencescreeningmethods★112ZhangCai-long,XiaChang-suo,JiangZh
2、eng-yaoAbstract1BACKGROUND:Underspecialconditions,bonemarrowmesenchymalstemcells(BMSCs)candifferentiateintoosteoblastsandDepartmentofchondroblasts.However,MSCsarefewinbonemarrow.Howtoharvest,purifyandrapidlyproliferateinvitroisafoundationofOrthopaedics,HospitalAffiliatedto
3、applicationintissueengineeringtechnique.MedicalCollege,OBJECTIVE:Tooptimize,collect,purify,assessrabbitBMSCsandtoobservethebiologicalcharacterofBMSCs.QingdaoUniversity,DESIGN,TIMEANDSETTING:TheobservationalstudywasperformedattheAnimalExperimentalCenterofTongjiMedicalQingda
4、o266003,CollegefromSeptember2005toJuly2006.ShandongProvince,2China;DepartmentMATERIALS:OnefemaleNewZealandrabbitsaged2monthswereusedforMSCcollectionandprimaryculture.ofPhysiology,METHODS:Bonemarrowsolutionwaspurifiedbydensitygradientcentrifugationandadherencescreeningmetho
5、d.CultureMedicalCollege,solutionwasobtained.BMSCswereincubatedinphosphatebufferedsolution(PBS),supplementedwith2.5g/Ltrypsin(3.0mL),QingdaoUniversity,andplacedinanincubatorat37℃fortwoorthreeminutes.Cellmorphologywasobservedusinganinvertedmicroscope.TheQingdao266021,Shandon
6、gProvince,digestionwasstoppedwhencytoplasmrecovery,longandthincellswithlargeintercellularspace,andfewroundcellsappeared.China8Subsequently,BMSCswereincubatedinserum-freeL-DMEM,andplacedinaplasticcultureflaskat1.0×10/L.MAINOUTCOMEMEASURES:MSCmorphology,ultrastructureandsurf
7、acemarker;Proliferationofthefirst,third,fifth,eighthZhangCai-long★,andtenthpassagesofBMSCs;Cellgrowthcurvewasdrawn.Master,DepartmentofOrthopaedics,RESULTS:BMSCswaspurefollowingdensitygradientcentrifugationandadherencescreeningmethod.ThethirdandfifthHospitalAffiliatedtopass
8、ageofcellshadtypicalwhirlpool-shape.Transmissionelectronmicroscopedemonstratedthatroundor
