大鼠脂肪基质细胞的分离培养与二步法诱导分化为神经细胞的实验.研究

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1、ABSTRACTAIM：Toisolate，purificateandamplifytheADSCsfromadultratsinvitroandtoobservetheirbiologicalproperties．METHODS：TocollecttheretroperitonealadiposetissueofSDratsunderasepticanestheticstate．Toshearitintopiecesandcentrifugefollowingtheenzymaticdigest

2、ionwithcollagenaseI，thencultivatethecellsinDMEM伊l2medium谢m10％offetalbovineserum(FBS)incultureflask37℃、5％C02．TotraceandinvestigatethegrowthconditionoflivingcellsundertheCK2contrastphasemicroscope．Todetectthecellularproliferationconditionbymethylthiazol

3、yltetrazolium(MTT)anddrawthecellulargrowthCHIVe．TodetectthecellularsurfacemarkerssuchasCD34、CD106、CD44andHLA一1、析mbothflowcytometerandimmunofluorescencecytochemicalstaining．RESULTS：Themajorityofinoculatedcellswereglobularfloatinginnutritivemediumandali

4、ttlelipiddropletsuspendingonthetoplevellayerundertheinvertedmicroscope．111epro-ADSCshaddiversemorphous，e．x．globular，longfusiformandfiberforming-like．nlecontaminatedcellswereeliminatedthroughtheadheringtransferofculture，andthecellularmorphousweremainta

5、inastheuniformity．Thelongfusiform，whirlpool—likecellslinedupinorderatthetimeof5mgeneration．mpassagecellsproliferatedtwiceinsizeper24handshowedstrongerchemicalpositivestainingofCD44byimmunocytochemistry．Theratesofcell-surfacemarkersundertheflowcytomete

6、rwereCD340．8％，CDl0613．6％，CD4499．7％，HLA．196．7％respectly，whichallconformedtothephenotypecharacteristicsofADSCs．CoNCLUSIONS：Itwasconvenientandfeasibletoadoptthemethodoftheisolation，civilizationofcells，inthisexperiment,theacquiredADSCscouldbehi曲lypurified

7、withthrivingcellularvigorandpowerfulself-renewalcapability．PartII：DifferentiationofadultratsADSCsintoneuralcellswithtwo-stepmethodAIM：ToinvestigatetheexperimentalmethodsofinducmentfromADSCsintoneurospheresthatconsistedofNSCs，andtheneuraldifferentiatio

8、nfromneurospheresintotheneuralcells．硕士学位论文METHODS：Weinducedanddifferentiatedpassage5ofADSCs，combined诵tllbasicfibroblastgrowthfactor(bFGF)20ng／ml，epithelialgrowthfactor(EGF)20ng／mlandN2(1：100)additives，intheserum-freeNeurobasalnutrientmedium．Th