简介：AIM:ToinvestigatetheroleofBrn-3bindifferentiationprocessofstemcellsderivedfromretinalMiillercellsintotheganglioncell.METHODS:ThepassageculturemethodofMiillercellsfromretinaofnewbornSpragueDawleyratswascarriedoutbyrepeatedincompletepancreaticenzymedigestionmethod.Thecellsweredetectedbyfluorescenceactivatedcellsorter(FACS),immunohistochemistrytechnologyandreversetranscription-polymerasechainreaction(RT-PCR)todeterminethepurity.Thethirdpassageofcellswasinducedintheserum-freededifferentiationmedium.TheexpressionofthespecificmarkersKi-67andnestinofretinalstemcellswasmeasuredbyRT-PCRandWesternblot.Thecellproliferationofretinalstemcellswasdetectedby5-Ethynyl-2’-deoxyuridine(Edu)staining.Thecellswererandomlydividedinto5groupsasfollows:groupA:Brn-3bsiRNAgroup;groupB:Brn-3bcontrolsiRNAgroup;groupC:pGC-Brn-3b-greenfluorescentprotein(GFP)group;groupD:pGC-GFPgroup;groupE:controlgroup(withoutanyhandling).ThepurifiedMullercellswereculturedfor3-7d,then,thepercentageofganglioncellswascountedbyimmunofluorescencestaining.RESULTS:FACSdemonstratedthepurityofretinalMullercellswasmore97.44%.Afewsphericalcellspheresappeared.Immunofluorescencestainingshowedthatstemcellswithinthesphereswerepositiveforretinalstemcell-specificmarkersnestin(redfluorescence,92.94%±6.48%)andKi-67(greenfluorescence,85.96%±6.04%).Meanwhile,RT-PCRanalysisshowedcellspheresintheculturetohaveexpressedabatteryoftranscriptscharacteristicofstemcellssuchasnestinandKi-67,whichwereabsentintheMullercells.WesternblotanalysisfurtherconfirmedtheexpressionofnestinandKi-67inthecellspheresbutnotintheMullercells.Edustainingshowedmostofthenucleiwithinthecellsphereswerestainedred(82.80%±6.65%),suggestingthenewcellsphereshadthecapacityforeffectiveproliferation.ThestatisticsresultshowedthedifferencebetweenBrn-3bsiRNAgroupand

简介：Excessivenoise,ototoxicdrugs,infections,autoimmunediseases,andagingcancauselossofspiralganglionneurons,leadingtopermanentsensorineuralhearinglossinmammals.Stemcellshavebeenconfirmedtobeabletodifferentiateintospiralganglionneurons.Littlehasbeenreportedonadiposetissue-derivedstemcells(ADSCs)forrepairofinjuredspiralganglionneurons.Inthisstudy,wehypothesizedthattransplantationofneuralinduced-humanADSCs(NI-hADSCs)canrepairtheinjuredspiralganglionneuronsinguineapigswithneomycin-inducedsensorineuralhearingloss.NI-hADSCswereinducedwithculturemediumcontainingbasicfibroblastgrowthfactorandforskolinandtheninjectedtotheinjuredcochleae.GuineapigsthatreceivedinjectionofHanks’balancedsaltsolutionintothecochleaewereusedascontrols.Hematoxylin-eosinstainingshowedthatat8weeksaftercelltransplantation,thenumberofsurvivingspiralganglionneuronsinthecelltransplantationgroupwassignificantlyincreasedthanthatinthecontrolgroup.Alsoat8weeksaftercelltransplantation,immunohistochemicalstainingshowedthatagreaternumberofNI-hADSCsinthespiralganglionsweredetectedinthecelltransplantationgroupthaninthecontrolgroup,andtheseNI-hADSCsexpressedneuronalmarkersneurofilamentproteinandmicrotubule-associatedprotein2.Within8weeksaftercelltransplantation,theguineapigsinthecelltransplantationgrouphadagraduallydecreasedauditorybrainstemresponsethreshold,whilethoseinthecontrolgrouphadalmostnoresponseto80dBofclicksorpuretoneburst.ThesefindingssuggestthatalargeamountofNI-hADSCsmigratedtothespiralganglions,survivedforaperiodoftime,repairedtheinjuredspiralganglioncells,andtherebycontributedtotherecoveryofsensorineuralhearinglossinguineapigs.