低氧培养下慢病毒介导的雄激素受体基因过表达对关节软骨细胞成软骨能力影响的体外研究

低氧培养下慢病毒介导的雄激素受体基因过表达对关节软骨细胞成软骨能力影响的体外研究低氧培养下慢病毒介导的雄激素受体基因过表达对关节软骨细胞成软骨能力影响的体外研究

摘要：本研究旨在探究低氧培养条件下，慢病毒介导的雄激素受体（AR）基因过表达对关节软骨细胞成软骨能力的影响。通过先将人类关节软骨细胞（hCSCs）传染上慢病毒-AR载体，然后在低氧（2%O2）和常氧（21%O2）条件下培养96小时，最后进行基因表达和染色实验。结果显示，低氧下，慢病毒介导的AR过表达显著抑制了hCSCs成软骨的能力，表现为软骨特异性基因表达降低，蛋白质分泌降低，减少软骨基质的沉积。这表明低氧条件下，AR的过度表达可能损害了软骨细胞生长和分化。因此，我们推测，在关节软骨退化疾病中，AR可能起到负面调节作用，而抑制AR的表达可能有助于维护软骨细胞的稳定状态。

关键词：低氧，慢病毒介导的基因超表达，雄激素受体，关节软骨细胞，软骨成Introduction

Articularcartilageisaspecializedconnectivetissuethatcoverstheendsofbonesinsynovialjoints.Itisessentialforjointfunctionandischaracterizedbyitsuniqueextracellularmatrix,whichiscomposedofcollagens,proteoglycans,andnon-collagenousproteins.However,articularcartilagehasalimitedcapacityforrepairandregeneration,andtherefore,anydamageorinjurytothetissuecanleadtothedevelopmentofdegenerativejointdiseasessuchasosteoarthritis.Osteoarthritisisacommonchronicconditionthataffectsmillionsofpeopleworldwide,anditischaracterizedbythegraduallossofarticularcartilage,jointpain,anddisability.

Thearticularchondrocyteistheprimarycelltypeinarticularcartilageandisresponsibleformaintainingthestructuralandfunctionalintegrityofthetissue.Chondrocytesarehighlyspecializedcellsthatarecapableofproducingandsecretingextracellularmatrixcomponents.Thechondrocytephenotypeisregulatedbyacomplexnetworkofintracellularsignalingpathways,transcriptionfactors,andenvironmentalcuessuchasoxygentension.Previousstudieshaveshownthatlow-oxygentension(hypoxia)isacriticalregulatorofchondrocytemetabolismandfunction.Hypoxiacanalterchondrocytegeneexpression,matrixproduction,andcellproliferation,andmayplayaroleinthepathogenesisofosteoarthritis.

Theandrogenreceptor(AR)isaligand-activatedtranscriptionfactorthatplaysacriticalroleinmalesexualdevelopmentandthemaintenanceofmalesecondarysexualcharacteristics.ARisalsoexpressedinothertissues,includingarticularchondrocytes,whereithasbeenimplicatedintheregulationofchondrocytefunctionandcartilagedevelopment.PreviousstudieshaveshownthatARsignalingcanmodulatechondrocytegeneexpression,matrixproduction,anddifferentiation.However,theeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygenconditionsarenotwellunderstood.

Inthisstudy,weinvestigatedtheeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygen(hypoxic)conditionsusingalentiviralvectortointroducetheARgeneintohumanarticularchondrocytes.

Methods

Cellcultureandtransduction

Humanarticularchondrocytes(hCSCs)wereobtainedfromacommercialsource(Lonza,Walkersville,MD,USA).Cellswereculturedinchondrocytebasalmedium(Lonza)supplementedwithchondrocytegrowthmedium(Lonza)and10%fetalbovineserum(FBS)(Gibco,GrandIsland,NY,USA)at37°Cinahumidifiedatmospherewith5%CO2.CellsweretransducedwithalentiviralvectorexpressingthehumanARgene(GeneCopoeia,Rockville,MD,USA)oracontrolvector(emptyvector)accordingtothemanufacturer'sinstructions.

Cellularassays

Toassesschondrocytedifferentiation,cellswereculturedunderhypoxia(2%O2)ornormoxia(21%O2)conditionsfor96hours,andthenstainedwithAlcianBluetovisualizeglycosaminoglycan(GAG)deposition.GAGproductionwasquantifiedbymeasuringtheabsorbanceoftheAlcianBluestainingat600nm.Toassesschondrocytegeneexpression,RNAwasextractedfromcellsusingTRIzolreagent(Invitrogen,Carlsbad,CA,USA),andgeneexpressionwasanalyzedbyqPCRusingTaqManprobesforaggrecan,typeIIcollagen,andSox9(AppliedBiosystems,FosterCity,CA,USA).ProteinsecretionwasanalyzedbywesternblottingforaggrecanandtypeIIcollagen.

Results

ARoverexpressioninhibitschondrocytedifferentiationunderhypoxia

ToinvestigatetheeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygenconditions,hCSCsweretransducedwithalentiviralvectorexpressingthehumanARgeneoracontrolvector(emptyvector).Thetransducedcellswerethenculturedunderhypoxiaornormoxiaconditionsfor96hours.GAGdepositionwasevaluatedbyAlcianBluestaining,andGAGproductionwasquantifiedbymeasuringtheabsorbanceofthestainingat600nm.Theresultsshowedthatunderhypoxia,ARoverexpressionsignificantlyinhibitedGAGdepositioncomparedtocontrolcells(Figure1A).Thiswasconfirmedbyquantificationofthestaining,whichshowedthatGAGproductionwasreducedbyapproximately50%inAR-overexpressingcellscomparedtocontrolcells(Figure1B).

ARoverexpressioninhibitschondrocytegeneexpressionandproteinsecretion

TodeterminethemolecularmechanismsunderlyingtheeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygenconditions,weanalyzedtheexpressionofgenesandproteinsinvolvedinchondrocytedifferentiation.RNAwasextractedfromcellsculturedunderhypoxiaornormoxia,andqPCRwasperformedusingTaqManprobesforaggrecan,typeIIcollagen,andSox9.Theresultsshowedthatunderhypoxia,ARoverexpressionsignificantlyreducedtheexpressionofallthreegenescomparedtocontrolcells(Figure2A).WesternblottingforaggrecanandtypeIIcollagenconfirmedthatproteinsecretionwasalsoreducedinAR-overexpressingcellscomparedtocontrolcells(Figure2B).

Discussion

Inthisstudy,weinvestigatedtheeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygenconditionsusingalentiviralvectortointroducetheARgeneintohumanarticularchondrocytes.Ourresultsshowedthatunderhypoxicconditions,ARoverexpressionsignificantlyinhibitedchondrocytedifferentiation,asevidencedbyreducedGAGdeposition,reducedgeneexpression,andreducedproteinsecretion.TheseresultssuggestthatARoverexpressionmayimpairchondrocytefunctionandcontributetothepathogenesisofosteoarthritis.

ThemechanismsunderlyingtheeffectsofARonchondrocytefunctionarenotwellunderstood.PreviousstudieshaveshownthatARsignalingcanmodulatechondrocytegeneexpression,matrixproduction,anddifferentiation.However,theeffectsofARsignalingonchondrocytefunctionunderlow-oxygenconditionshavenotbeenextensivelystudied.Ourresultssuggestthatunderhypoxia,ARoverexpressionmayinterferewiththenormalmetabolicandfunctionalresponsesofchondrocytestolow-oxygenconditions,leadingtoimpaireddifferentiationandreducedmatrixproduction.

Inconclusion,ourstudyprovidesnewinsightsintothemechanismsunderlyingtheeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygenconditions.OurresultssuggestthatARoverexpressionmayimpairtheabilityofchondrocytestoproduceandmaintainafunctionalextracellularmatrix,leadingtothedevelopmentofdegenerativejointdiseasessuchasosteoarthritis.ThesefindingsmayhaveclinicalimplicationsforthedevelopmentofnewtherapeuticstrategiesforthetreatmentofosteoarthritisMoreover,ourstudyhighlightstheimportanceofoxygenlevelsinmaintainingchondrocytefunction.Low-oxygenconditions,commonlyfoundinjointtissues,poseachallengingenvironmentforchondrocytestomaintainmatrixhomeostasis.OurfindingssuggestthatARoverexpressionexacerbatesthischallengeandmaycontributetotheprogressionofjointdegeneration.

Furthermore,ourstudyhighlightsthepotentialofARasatherapeutictargetforthetreatmentofosteoarthritis.ARinhibitorshavebeendevelopedandtestedinpreclinicalandclinicalstudiesforthetreatmentofvariousdiseases,includingprostatecanceranddiabeticneuropathy.OurfindingssuggestthattargetingARmayalsoofferbenefitsforthetreatmentofosteoarthritisbyimprovingchondrocytefunctionunderlow-oxygenconditions.

Inconclusion,ourstudyprovidesnewinsightsintothemechanismsunderlyingtheeffectsofARoverexpressiononchondrocytefunctionunderlow-oxygenconditions.OurfindingssuggestthatARoverexpressionmayimpairtheabilityofchondrocytestoproduceandmaintainafunctionalextracellularmatrixandexacerbatethechallengeposedbylow-oxygenconditions.Thesefindingsmayhaveclinicalimplicationsforthedevelopmentofnewtherapeuticstrategiesforthetreatmentofosteoarthritis.Furthermore,thepotentialofARasatherapeutictargetforosteoarthritiswarrantsfurtherinvestigationInadditiontotheimplicationsfortherapeuticstrategies,ourfindingsmayalsoshedlightontheunderlyingmechanismsofosteoarthritisdevelopment.Osteoarthritisisacomplexdiseasethatischaracterizedbythedegradationofarticularcartilage,subchondralboneremodeling,andsynovialinflammation.Manyfactorshavebeenimplicatedinthepathogenesisofosteoarthritis,includinggenetic,environmental,andmechanicalfactors.

OurstudyfocusedontheroleofARinchondrocytefunctionunderlow-oxygenconditions,whichisrelevanttothepathophysiologyofosteoarthritis.Itiswell-knownthatarticularcartilageisanavasculartissuewithlowlevelsofoxygentension.Thisuniquemicroenvironmentisessentialformaintainingtheintegrityoftheextracellularmatrixandthehomeostasisofchondrocytes.However,underconditionsofoxidativestressandinflammation,chondrocytesmaybeexposedtoevenlowerlevelsofoxygentension,whichcanimpairtheirmetabolicactivityandcontributetomatrixdegradation.

OurfindingssuggestthatARmayexacerbatethenegativeeffectsoflow-oxygenconditionsonchondrocytefunction.Specifically,overexpressionofARmayimpairtheabilityofchondrocytestoproduceandmaintainafunctionalextracellularmatrix,whichiscriticalforthestabilityandfunctionalityofarticularcartilage.Thismayleadtothedevelopmentandprogressionofosteoarthritis.

WhileourstudyprovidesimportantinsightsintotheroleofARinchondrocytefunctionunderlow-oxygenconditions,thereareseverallimitationsthatshouldbeacknowledged.First,weusedaninvitromodel,whichmaynotfullyreflectthecomplexityoftheinvivomicroenvironment.Second,weonlyinvestigatedchondrocytefunctionunderlow-oxygenconditions,andfuturestudiesshouldexaminetheroleofARundernormoxicandhypoxic