【doc】雌二醇与孕激素对人成骨细胞护骨素基因作用的比较

【doc】雌二醇与孕激素对人成骨细胞护骨素基因作用的比较 雌二醇与孕激素对人成骨细胞护骨素基因 作用的比较 中国组织研究与临床康复第,,卷第10朋2007-03-11出版 JournalofClinicalRehabil#at/veTissueEngineeringResearchMarch11,20 07VoL11,No.10 BasicMedicine 17beta..estradiolversusprogesteroneintheexpressionof osteoprotegeringeneinhumanosteoblast--likecells Ouyang]unI,LiaoEr—yuan,LuoXiang—hang,ShaoHui,ge,ZhouHou—d e ‘Departmentof Endocdnok>gy.First HospitalofChangsha C时.Changsha 410005.Hunan Provi~.China; Zlnst~tuteof Endocrinologyand Metabolism.Second XiangyaHospitalof CentraISouth University.Changsha 410011.Hunan Province.China OuyangJun. Associatechief physician.Department ofEndocdno~gy.First HospitalofChangsha City.Changsha 410005.Hunan Provi~China xianghangluo@ 21cn.ODIn ColTespocldenc~to: LuoXiang-hang. Doctor,Professor. Inamuteof Endoodnok>gyand Metabolism.Second XiangyaHospitalof CentraISouth Univemity,Changsha 410011Hunan Province.China Received:20o昏10.11 Accepted:2006-11-14 (06-50-6-4949/W) OuyangJ.LiaoEY. LuoXH.ShaoHG. ZhouHD17 beta-estTadiolversus progesteroneinthe expressionof osteoprotegenngene inhuman osteoblast4ikecells. ZhongguoZuzhi GongchengYanjiuyu UnchuangKangfu 2007;11(1O):1976- 1979fChina) [w.zg~kf.com/ zgIckf/ejoumal/ upnles/07-10/ 10k-1976(ps).pdq 1976 Abstract BACKGROUND:Estrogen/progestinsreplacementtherapypreventsexcessbonelossinpostmenopausalwomen.’ Recentlyosteopmtegerin(OPG)hasbeenidentifiedinosteoblastanddisplayedtoinhibitboneresorption. OBJECTIVE:Tocomparetheactionbetween1713-estradiol(E2)andprogesteroneonOPGexpressionincultured normalhumanosteoblast—likecells(hOB). DESIGN:Acomparativeinvestigation SETFING:InstituteofMetabolicEndocrinology,theSecondXiangyaHospitalofCentralSouthUniversity MATERIALS:n—MEM(SigmaChemicalCorp.,St.Louis,MO,USA);TypeIVcollagenase(Sigma);Fetalbovineserum (Gibco-BRLCorp.,GrandIsland,NY,USA);Osteocalcinradioimmunoassaykit(DiaSorinCorp.,Stillwater,MN,USA). METHODS:TheexperimentswerecardedoutintheInstituteofMetabolicEndocrinology,SecondXiangyaHospitalof CentralSouthUniversityfmmJanuary2003toMarch2006..Theosteoblasts wereextractedfr0mthecancelousboneof anteriorsuperioriliacspineofnormalpeople,thencultured.ThehOBweretreatedwithE2andprogesterone,andthe expressionsofOPGmRNAandOPGproteinweredeterminedbyNorthemblotanalysisandenzyme’linked immunoabsorbentassay(ELISA)respectively. MAINOUTCOMEMEASURES:~Characterizationofhumanosteoblast—likecells;?EffectofE2andprogesteroneonOPG mRNAlevelsbyNorthemblotanalysis;? EffectofE2andprogesteroneonOPGproteinlevelsintheconditioned mediumbyELISA. RESULTS:? CharacterizationofhOBinvitro.TheALPIevelsinnormaIhumanosteoblastswere(74.3+4.7)U/gprotein, andthedetectableosteocalcinIevelswas(3.84_+0.39)旧,Lprotein],whichsuggestedthatosteoblastsweretheprimary celItypefoundinourbone-derivedcelIculturesfromdonors.(EffectsofE,andprogesteroneontheIevelsofOPG mRNAbvNorthemblotanalysis:TheOPGmRNAbandwasweakinthecontroIgroup【(12.3?3.5)%l,treatmentwith1× 101×10.9,1×10moPLE2causedanincreaseintheIevelsofOPGmRNA.TheexpressionofOPlGmRNAinthe1× 10mol/LE2groupwasgraduallyincreasedat12,24and48hours.ProgesteronehadnoinfluenceonOPGmRNA expression.(EffectsofE,andprogesteroneonOPGproteinproductioninconditionedmediumdeterminedwithELISA: ELISArevealedthattreatmentwith1×10I’1×10.9,1×10mol/LE2inducedobviousincreaseintheIevelsofOPG proteinincellsmediaascomparedwiththatinthecontroIgroupf(1.27?0.26),(2.34?0.35),(3.62?0.23),(0.64?0.14) ‘Lg,L,P<0.011.Inthepresenceof1×10moPLE2IOPGproteinproductionincellsmediaat12,24and48hourswere significantlyhigherthanthatinthecontrolgroup 【(1.30_+0.30),(3.07_+0.14),(3.50_+0.33),(0.62_+0.12)g,L,P<0.011.1× 10I’o,1×10.9,1×10.8mol/LprogesteronehadnoinfluenceontheOPGproteinproductionafter12—24hours(P>0.05). CONCLUSION:ThedifferentregulationofOPGproductioninosteoblastsbyE2andprogesteronemaycontributetothe mechanismsbywhichestrogenorprogestinsexertsitsdifferentactiononboneresorption. _NTRoDUCT_oN Theimportanceofsexsteroidsinthemaintenanceof bonemasshasbeenwidelyacceptedr~i.Inpostmenopausal exogenousestrogenandprogestinscan preservebonemass.Estrogenreceptorandprogesterone receptorhavebeencharacterizedinosteoblast{21.The effectsofestrogenandprogestinsonbonemetabolism arebelievedtopartlybemediatedbynuclearsteroid receptorsI’?. Osteoprotegerin(OPG,alsoknownasosteoclastogenesis inhibltoryfactor),producedbyosteoblasts,isanegative regulatorofosteoclastformationn41.Thisfactorfunctions asadecoyroceptorforreceptoractivatorofnudear fa~tor-KBligand(RANKL,alsoknownasosteoclast drentiationfactor/osteoprotegerinligand/tumornecrosis factor-relatedactivation—inducedcytokine1andinhiblts osteoclastformationbyinterruptingtheRANKL.RANK interactionbetweenosteoblastandosteclastprogenitors12-4]. OPGispresentedasaheparin.bindingsecretory glycoprotein[51.OPGknockoutmicedevelopsevere osteoporosisandhaveincreasedosteoclastogenesis, markedboneIOSS,destructionoftheirgrowthplates,and Iacktrabecularboneintheirlongbones[0,7i.OPGprevents boneIOSSinovariectomizedratsandincreasesbone mineraIdensityandbonevolumeinnormaIrats【8】. Furthermore,ithasbeendemonstratedthatestrogen stimulatedOPGproductioninhumanosteoblasticcells[9l, and17beta-estradioI(E2)promotedtheexpressionof OPGbybonemarrowstromallineI’.whiletheeffectof progesteroneonOPGproduction;nosteoblastis _, unknown. Thepresentstudywasundertakentoinvestigateand comparethee仟ects0fandproaester0ne0nOPG expressi0ninn0rmaIhuman0ste0bIast.Ijkece?sfhOB) cunlJred加咖s0ast0exp10rethemechanism0fE2 andp巾gester0nein陀guIatingbonemetaboIism. 嚣;薯第kfc23N38’5.~’53sina.com欧阳俊,等.雌二醇与孕激素对人成 骨细胞护骨素基因作用的比较II,I’Iz眦艋.欧阳俊,等.雌二醇与孕激素 对人成骨绌胞护骨黍基凼作用围比 MATER?ALSANDMETHoDS Materials TheexperimentswerecarrtedoutintheInstituteofMetabOIic Endocrinology.SecondXiangyaHospitalofCentralSouthUniversity fromJanuary2003toMarch2006.TypeIVcollagenase(Sigma),fetal bovineserumfGibco-BRLCorp.,GrandIsland,NY,USA),MEM (Sigma).OPGradioimmunoassaykit(DiaSorinCorp.,Stillwater,MN, USA1. Methods CellCultures BonesampJeswereObtainedwithjnformedconsentfromdonorsafter approvedbytheLocaIResearchEthicsCommittee.Primarycultures ofnormaIhOBwerepreparedfromtrabecularboneobtainedduring surgeryforroadtrafficaccidentvictims(agerange35-55years),as previouslydescribed删.NoneofthebonedonorshadclinicaI symptomsorhistoryofbonemetabolicdisordsis.Briefly,surgical specimensofribwererinsedextensivelywithserum-freea-MEM.The trabacularboneswereharvestedwithaSize00curette.minced,cut intosmalIpiecesof1mmx1mmx2mm.andthenwashedforseveraI timestoremovebonemarrow.Thebonechipsthusobtainedwere incubatedwithtype?coilagenase(1g,L)for2hoursat37?,and thedigestionwasterminatedwithphenolred-freed-MEMcontaining FBSf0.2involumefraction).Thedigestedchipswerewashed extensivelytoremovereleasedcellsanddebris.Chipswerecultured in25cmflasksinphenolred-freed-MEMcontainingFBSf0.15in volumefraction).100U,mLpenicillin.100mg,Lstreptomycin,and 50mg,Lascorbicacidat37?,theculturemediumwaschanged after2days,thenonceevery3days.After15days,cellsmigrated fromtheboneparticlesandreachedconfluenceafter25days.They werethenreleasedfromthebonechipswith2.5g/Ltrypsin.EDTA (Sigma),countedandsubculturedat105cells/bottlein25cmflasksin phenolred-freeo~-MEMcontainingFBS.100U,mLpenicillin.100mg,L streptomycin,and50mg,Lascorbicacidandreachedconfluence after7days. Interventiontests Intheexperimentsofcellstreatmentwith17~-estradiol-watersoluble (Sigma)orprogesterone-watersoluble(Sigma).hOBwerePIated.n 25cmflaskinphenolred-freed-MEMcontainingFBSf0.1in volumefraction),100U,mLpenicillin.100mg,Lstreptomycin,and 50mg,Lascorbicacid.Afterreachedconfluence,thecellswerethen culturedinphenolred-freed-MEMcontainingFBSf0.01involume fraction1for4days.Cellsweresubsequentlyincubatedf0r48hours inphenolred-freed-MEMcontaining2.5g/Lbovineserumalbumin (Sigma).ThecellsweretreatedwithvehicleorE2at1×100,1×10-9, 1×10r8mol/L,progesteroneat1×101×10_9.1xl0-8mol/Lfor48 hoursinphenolred-freed-MEMcontaining2.5g/Lbovineserum albumin.Cultureswerealsoexposedtofreshserum-freemedium withorwithout1×10-8moULE2Or1×10-8mol/Lprogesteronefor12— 48hours.ConditionedmediaofhOBcultureswerecollectedand storedfrozenat一70?untiIassayedbyenzyme-linked immunoabsorbentassayfELISA).T0taIRNAwasextractedfmthe celJlayersforNorthemblotanalysis.ThecelIslayerswerealso harvestedfortotaIproteindetermination. HoBweregrowntoconfluenceinthe25cm0flasksordishes (60-rnm)asdescribedabove.Thecellswerewashedfor3timeswith PBS,andthenthecellslaIyerswerescrapedintothesolution containing20mmol/LTris-HCI.pH8.0.and150mmol/LNaCI,1% TritonX-1o0.0.02%NaN3.1mg/Laprotinin.Thelysatewere homogenizedbysonicationfor20s.ThentheactMtyofalkaline phosphatase(At.P)wasmeasuredin3differentflasksderivedfrom 沈阳1200邮政信箱110004kf23385083@sina.cornw.zgldd.mm eachcultureusinganALPkitfSigma). Osteocalcinreleasedintotheculturemediawasmeasuredin triplicateusingaspec~cradioimmunoassaykjt.TOnormalizethe proteinexpressiontototaIcellularprotein,afractionofthelysate solutionwasusedinaBradfordproteinassay. Cellsindis~swerestimulatedfor20minuteswith1xl0-7mol/L humanparathyroidhormone(1—34)(PTH1—34,Sigma).andcyclic adenosinemonophosphate(cAMP)wasmeasuredafter trichloroaceticacidprecipitationofthecelIextractsusingacAMP assaykit(ChinaatomicenergyInstitute.China).Theresultswere expressedaspicomolesofcamppermilligramsofcelIprotein. MeasurementofOPGmRNAlevelsbyNorthernblot analysis T0laIRNAwasisolatedusingtrizolreagent(Gibco)accordingtothe recommendedprotoco1.DNAprobeforOPGweregeneratedby reversetranscription(RT)-polymerasechainreaction(PCR)withthe totaIRNAfr0mhOBaspreviouslydescribedis,”.RTwasperformed using1.0gtotaIRNAandthereversetranscriptionsystem (Promega,Madison,WI,USA).ForOPG,complementaryDNA (cDNA).thePCRprimerswere5’-芦CCCCAGAGCGAAATAC-3’ and5’.AAGAATGCCTCCTCACAC.3’.yieIdinga219-basepair (bp)fragment.AfterRT,PCRwasperformedasfoIlowad:94?for 1minute.56?f0r1minute.and72?for1.5minutesfor35cycles followedbya10.minuteincubationat72?.TheidentitiesofPCR productswereconfirmedbydirectsequencingusinganautomatic DNAsequence(PEAppliedBiosystems,Norwalk,CT.USA).An 1100-bpGlyceraldehyde-3-phoshatedehydrogenase(G3PDH)cDNA waspurchasedfromCIontechLaboratoriesInc.ThecDNAfragments wereradiolabledwiththerandomprimersDNAlabelingsystem (Gibco)and【dCTP(BeijingatomicenergyInstitute,China). Samplesof40p.gtotaIRNAwereseparatedbyelectrophoresisin formaldehyde(10g,L)agrosegelsandtransferredtoaHybondN+ nylonmembranefAmershamPharmacia).Hybridizationswerecar—ed outat42?f0r24hourswitha32P-labeledOPGcDNAD?be.Fine werewashed,『I,ith0.1×SSC(150mm0NaCI.15mmo虬sod.um cit阳te)and1%SDSat37?.ThebandswerevisuaIiz,}dbv aut0阳diOgraphyinrefrige阳t0rat一70?眦,anddeIem1inedbv densit0metry.OPGmRNAIeveIswerenOm1a?zedbyG3PDH rep?bingthesameb10t.NonhembanaIysessh0vvnwere repreSema埘veofthejndependemcunus. M?srementofoPGp?tein mediumbyELlsA Ce?supematantsmediawere?IIec【edf0rmeasurement0fthe amounts0fOPG,『I,ithELlSAk.ts(BioVendorlnc.).Ac?rdingt0the instnJdiOns.theamountsOfOPGpr0teinpresentedinthecOnditiOned med.awemeasured. Statisticalanalyses A?experimentswerepeatedf0ratIeastth怕etimes,thedatawere presentedasMean?SD.COmparisOns登madeby0ne-way anaIysisofVananceandranksumtest.and怕presentative exDerimentsaresh0wn. RESULTS Cfhumanosteob?ast.?Ikece?sIhoB)jh vitm CeIIsObta.nedf?mhumantrabecuIarbonewerecharadenzedas OsteobIast.IikeOe?sbyseveraIcritenaincIudinghighintrinsicALP adiv时.sec怕tionofosteocaIdn.andcAMPsponset0PTH.ALP IeveIsinnom1aIhumanosteobIastswere(74.3?4.7)U,gpr0tein. FdiOimmunoassay0fcuItu怕supema1antsf?munstjmuIatedhuman bonece?scunu陀sreveaIedde1edabIeOsteocaIcinIeveIs『f3.84? 0.39)U,gp彤in】.-Th?ereSuItsshowedthatosteobIastswe陀the ISSN1673.8225CN21-153g『R 欧阳俊,等.雌二醇与孕激素对人成骨细胞护骨素基因作用的比 较,~w.zg/c/cfcornkf23,~na,com primarycelltypefoundinourbone-derivedcellculturesf巾mdonors? EffectsofE2andprogesteroneonthelevelsofOPG mRNA TheOPGmRNAbandwasweakinthecontrolgroup【(12.3+3.5)%】, treatmentwith1×10-’1×10.1xl04mol/LE2causedanincreasein thelevelsofOPGmRNA【(33?4)%.(48+10)%,(77+13)%.P<0.001】 (Figure1).AsindicatedinFigure2.theexpressionofOPGmRNAin the1xl0mol/LE2groupwas(12?4)%,anditwassignificantly increasedafter12.24and48hours.TheresultssuggestedthatE2 increasedtheIevelsofOPGmRNAindose-andtime-dependent manRers. Figure1showedthattheintensitiesofOPGmRNAexpressionwere equivalentbetweenthehOBcontroIgroupandthe1×10-’1×10.1x 10_8mol/Lprogesteronetreatedgroups【(13?5)%.(12?6)%.(11?5)% and(14?7)%.P>0.05].Figure2showedthattheintensitiesofOPG mRNAexpressionaftertreatmentwith1×10.8mol/Lprogesteronefor 0.12.24and48hourswerenotsignificantlydifferent【(12?3)%,(13? 4)%,(12?7)%.(12?5)%.P>0.05].Itwasindicatedthatprogesterone hadnoinfluenceonOPGmRNAexpression. EffectsofE2andprogesteroneonOPGprotein productioninconditionedmediumdeterminedwith EL?SA ELIS;Arevealedthattreatmentwith1xl0-’o.1×10,1×10.8mol/LE2 inducedobviousincreaseintheIevelsofOPGproteinincellsmedia ascomparedwiththatinthecontrolgroup【(1.27+0.26),(2.34+0.35). (3.62i-0.7.3)LP<0.01].Inthepresenceof1×10r8mol/LOPG proteinproductionincellsmediaat12.24and48hourswere significantlyhigherthanthatinthecontrolgroup[(1.30i-0.30),(3.07i-0.14). (50?0.33).(0.62i-0.12)旧,L,P<0.01].1×101×101×10mol/L progesteronehadnoinfluenceontheOPGproteinproduction【(0.62+ 0.1o).(o.67+0.16),(0.62+0.15)帽,L,P>0.05].Inthepresenceof1x 10.8mol/Lprogesterone.OPGproteinproductionincellsmediaat12. 24and48hourshadnodiffeFenceascomparedwiththatinthe controlgroup[(0.58?0.12).(0.61?0.20),(0.62+0.18)L.P>0.05]. TheresultssuggestedthatE2increasedtheOPGproteinproduction 1978 ndose-andtime-dependentmanners,whereasprogesteronehadno nfluenceonOPGproteinproduction. DlSCUSSloN ThepresentinvestigationwasundertakentodeterminewhetherE2or progesteroneregulatesOPGexpressioninculturesofnormaIhuman osteoblasts(hOB).Thesestudiesdemonstratedthatthesynthesis andsecretionofOPGinosteoblastwereup-regulatedbyE2.butnot byprogesterone. TheOPGproteinsecretionandmRNAIevelsinculturesofhOB wereexamined.Itwasfoundthatafter12-48hoursoftreatment.E2 al1x10-8mol/LincreasedOPGmRNAIevelsandproteinproduction inculturesofhOB.treatmentwithincreasingdoseofE2causeda dose-dependentincreaseintheexpressionofOPGmRNAand proteinbyhOB.However.theresultsalsorevealedthatafter12—48 hoursoftreatment.progesteroneat1×10.8mol/Lhadnoeffectson OPGmRNAIevelsandproteinproductioninculturesofhOB.and treatmentwithincreasingdoseofprogesteronedidnotregulatethe expressionofOPGmRNAandproteinbVhOB. OPGhasrecentlybeenidentifiedasasolublememberofthe TNF.Rfamily[3-5].Thatactsasaparacrinefactorwithinthebone microenvironmenttodecreaseboneresorption.OPG.islikelytobe amajorregulatorofbonemetabolismbecauseitisproducedby os(eoblasts.anditsproductionisregulatedbymanyofthemajor calcitropichormonesandcytokines,suchasestrogen,1. 25-dihydroxyvitaminD3,parathyroidhormone.TNF-~.intedeukin-1 betafIL.113)i9].OPGisapotentinhibitorofosteoclastformation andactivation[3-5].OPGadministratedtonormaIratsincreasesbone densitybyinh?