搜索
:(973)(2005CB522604);(30425023);(30530410):400042,,:,Email:linchen70@163.com:R32217+1:A:100627108(2008)0320159205:,24h48,24h(tartrate2resistantacidphosphatase,TRAP),RT2PCR29(MMP29)TRAPK(CathepsinK)5TRAP(+),13TRAP(+);7,,;TRAP3,MMP29CathepsinK5,,:;;Establishmentofco2culturemodelofmouseosteoblastsandosteoclastsLUXiumin,CHENLin,SUNan,etal.StateKeyLaboratoryofTrauma,BurnsandCombinedInjury,TraumaCenter,InstituteofSurgeryResearch,DapingHospital,ThirdMilitaryMedicalUniversity,Chongqing400042,ChinaAbstract:ObjectiveToestablishaco2culturesystemofmouseosteoblastsandosteoclastsforfurtherinvestigatingtheinteractionfunctionofthem.MethodsPrimaryosteoblastsandosteoclastsareobtainedrespectivelyfromthecalvariasofnewbornmiceandlongbones(femurandtibia)ofadultmice(428weeksold).Anindirectcontactco2culturesystemissetupbyinsertingaglasssheetstickingbonemarrowmononuclearphagocytesintoculturedishwhichhadinoculatedosteoblastsfor24h.Theresistantacidphosphatase(TRAP)stainingandboneresorptionessaywereusedtoidentifytheosteoclastsafter3daysofco2culture.Themarkedgenesofosteoclast,MMP29,TRAPandCathepsinK,weredetectedbyRT2PCR.ResultsTheTRAPpositivemultinuclearcellsemergedafter5daysofco2cultureandthenumberofTRAPpositivemultinuclearcellsreachedthemaximumafter13days.Boneresorptionessayindicatedthatresorptionpitsformedonboneslicesafter7daysofco2cultureandtheareaofpitsgraduallyincreasedwiththeperiodofco2cultureextending.ThemarkedgeneofosteoclastofTRAPexpressedafter3daysofco2culture,buttheMMP29andCathepsinKweredetectedafter5days.ConclusionsTheco2culturesystemcaninduceosteoclastswhichdegradedboneactively.Thismodelcanprovidefoundationfortheresearchontheinteractionfunctionofosteoblastsandosteoclasts.Keywords:Osteoclast;Osteoblast;Co2culture,P(receptoractivatorofnuclearfactor2Bligand,RANKL)(macrophagecolonystimulatingfactor,M2CSF)95120083143ChinJOsteoporos,March2008,Vol14,No13,,111148SPFC57,24h,1122MEM(Gibco);(Gibco);0125%(Hyclone);RNA(Genemega);(TaKaRa);(Sigma);(TRAP)(Sigma)113200m,5mm5mm,50m,3,10min1000UPml1000UPmlD2Hanks,3,30min,2MEM114Vicky[1],24h,75%5min,,;PBS3,0125%,3720min,;PBS3,011%,371h,,200,1000rPmin,5min,DMEM;2180%,335mm115Takahashi[2],48,,,1ml2MEM(100UPml100UPml,2mmolPLL210%),,200100mm,37,5%CO2,95%24h(),1000rPmin,5min,2MEM,5106Pml011%24mm24mm5mm5mm11624hPBS,,24h35mm,,121,,111711711:11712TRAP:3579111315TRAP,,TRAP(+)(33),6(40),,P11713:3579111315,[3],215%410min,50kHz5min,,,1%34min,,(,),SPOTRTcameraPsoftware,311714:35791113,GenemegaRNA,DNaseDNA,RT2PCRMMP29CathepsinKTRAP,2actin(BandScan510)1,PCR:94,30s;58,30s;72,106120083143ChinJOsteoporos,March2008,Vol14,No13min;281MMP29(162bp)F:52CCTGTGTGTTCCCGTTCATCT23R:52ACCCGAATCTAGTAAGGTCGC23CathepsinK(140bp)F:52GGAAGAAGACTCACCAGAAGC23R:52GACACCTCCGCCGATATATCG23TRAP(240bp)F:52GCTGGAAACCATGATCACCT23R:52TTGAGCCAGGACAGCTGAGT232actin(738bp)F:52TTGTTACCAACTGGGACGACATGG23R:52GATCTTGATCTTCATGGTGCTAGG2311715:(xs),SPSS1010t,P01052211,5,,,,,,,35,(2)21221211TRAP:(TRAP),,,TRAP,5TRAP(+),9TRAP(+),,30;,TRAP(+)(P0105);15,TRAP(+),,TRAP(+),,(34)21212:,,,1%2(200)A1B.93TRAP(40)A1B.916120083143ChinJOsteoporos,March2008,Vol14,No134TRAP7,,(P0105);,,,(56)51%(200)A:B:921213:,29(MMP29)6K(CathepsinK)TRAP,RT2PCR,3TRAP,MMP29CathepsinK5d(7)73,P,,,,Takahashi[4]15,Sandra[5],[6],1,25(OH)2D3IL2126120083143ChinJOsteoporos,March2008,Vol14,No13,,Peter[7]24,,,[8]6,,,,,Matthias[9]Transwell24,,,Peter,24h,,:,,,,RNA,,,,,,[1]KartsogiannisV,NgKW.Celllinesandprimarycellculturesinthestudyofbonecellbiology.MolecularandCellularEndocrinology,2004,228:792102.[2]TakahashiN,UdagawaN,TanakaS,etal.BoneResearchProtocols.MethodsinMolecularMedicine,2005,80:1292144.[3]..:,2001,68269.[4]TakahashiN,AkatsuT,UdagawaN,etal.Osteoblasticcellsareinvolvedinosteoclastformation.Endocrinology,1988,123:260022602.[5]SandraZ,Orlandini,LuciaF,etal.Functionalandstructuralinteractionsbetweenosteoblasticandpreosteoclasticcellsinvitro.CellTissueResearch,1995,281(1):33242.[6],LorenzoJ.1,25(OH)2D31mRNA.,1998,33(6):3752377.[7]PeterML,RichardPE,HowardCT,etal.Osteogenicandosteoclasticcellinteraction:developmentofaco2culturesystem.CellTissueResearch,1998,294(2):992101.[8],,,..,2006,4(2):68269.[9]KieslingerM,FolberthS,DobrevaG,etal.EBF2Regulatesosteoblast2dependentdifferentiationofosteoclasts.DevelopmentalCell,2005,9:7572767.(:2007209203)(193),,,,,[1]CompstonJE,PapapoulosSE,BalanchardF.ReportonosteoporosisintheEuropeanCommunity:currentstatusandrecommendationsforfucture.WorkingPartyfromEuropeanUnionMemberStates.OsteoporosisInt,1998,8(6):5312534.[2]..,2003,6(2):67268.(:2007207228)36120083143ChinJOsteoporos,March2008,Vol14,No13