農(nóng)學代謝性骨病分子細胞生物學

農(nóng)學代謝性骨病分子細胞生物學第1頁/共49頁代謝性骨病代謝性骨病是一類伴有鈣、磷、維生素D代謝失調(diào)以及甲狀旁腺功能、細胞因子與激素分泌異常的全身性骨骼病

[代謝性骨病的分類]

1.骨質(zhì)疏松

2.佝僂-軟骨病

3.原發(fā)性甲狀旁腺機能亢進癥

4.原發(fā)性甲狀旁腺機能減退癥

5.中毒性骨病(維生素D、氟、鎘、鉛、磷等中毒)

6.其他如粘多糖代謝異常，Marfan癥，Paget癥,SEDT-PA。osteoporosis：Representsacontinuum,inwhichmultiplepathogeneticmechanismsconvergetocauselossofbonemassandmicroarchitecturaldeteriorationofskeletalstructure.

Thesefactors,coupledwithanincreasedriskoffalls,contributetoahighincidenceoffragilityfracturesinosteoporoticpatients.第2頁/共49頁Skeletalfragilitycanresultfrom:(a)failuretoproduceaskeletonofoptimalmassandstrengthduringgrowth;(b)excessiveboneresorptionresultingindecreasedbonemassandmicroarchitecturaldeteriorationoftheskeleton;(c)aninadequateformationresponsetoincreasedresorptionduringboneremodeling.第3頁/共49頁Theboneremodelingunitsorbonemulticellular

units(BMUs)

第4頁/共49頁骨組織的細胞成份成骨譜系：間充質(zhì)干細胞，骨源性始組細胞（osteoprogenitorcells）、成骨細胞（osteoblast）、骨襯細胞（bone-liningcell）、骨細胞（osteocyte）破骨譜系：多潛能造血干細胞、破骨細胞（osteoclast）成軟骨細胞、軟骨細胞、破軟骨細胞InstituteofMetabolism&Endocrinology,CentralSouthUniversity第5頁/共49頁非細胞成分膠原非膠原蛋白骨礦物質(zhì)血管神經(jīng)InstituteofMetabolism&Endocrinology,CentralSouthUniversity第6頁/共49頁組織細胞學基礎InstituteofMetabolism&Endocrinology,CentralSouthUniversityOsteoblastdevelopmentanddifferentiation第7頁/共49頁TranscriptionfactorsCbfa1/Runx2++++++?SurfacemarkersSTRO-1+++/----SB-10/ALCAM00----HOP-26/CD63++----EnzymesAlkalinephosphatase---++++++-Phex----++++Collagenandnon-collagenousproteinsCollagentypeI?-+++++++-Osteocalcin-----+++-Bonesialoprotein--++-+++++++++Osteopontin+?-/+-/+-/+-+++++++第8頁/共49頁EffectsofWntsignalingonosseouscells.ThecanonicalWntsignalingpathwaypromotestheproliferation,expansionandsurvivalofpre-andimmatureosteoblasts.Dkks,Sfrps,andWif-1antagonizeWntsignalinginosteoblaststofacilitatedeathofimmaturecells,buttheymayalsodownregulatethepathwayinmaturecellstoinduceterminaldifferentiation.第9頁/共49頁Wntsignalinginosteoblastsandbonediseases第10頁/共49頁OsteoclastdevelopmentanddifferentiationNormaldifferentiationrequires:

tyrosinekinase-andtumornecrosis-familyreceptors,normallyfmsandRANK.Ligandsforthesereceptorsplusunidentifedserumorcell-presentedfactor(s)areneededforinvitrodifferentiation,possiblysignallingviaanimmune-liketyrosinekinaseacceptormolecule.Osteoclastdevelopmentandactivity:increasedbycytokinessignallingthroughGP130,suchasIL-6,byTGF-b,andbyIL-1.Othertyrosinekinasereceptorsincludingkitandmetcanaugmentfmssignalling,andTNFsotherthanRANKL,includingTNFaandTRAIL,modifyRANKsignalling.ThesituationisfurthercomplicatedbyG-proteincoupledreceptorsincludingthecalcitoninreceptor,byintegrinorcalcium-mediatedsignals,andbyestrogenreceptors,whichoperateinbonelargelyviaNOdownstreamsignals.Differentiation,activity,andsurvivalsignalsmergeinintracellularsecondmessengers:includecytoplasmickinasesofseveralfamilies;differentiationpathwaysoftenterminateinErk/JunkinasesorNF-jB.KeyregulatoryintermediatesincludeTRAF6,src,Smad3,phosphatidylinositol-3-kinase,Jak/Stat,andthecGMP-dependentproteinkinaseI.Therearesubstantialuncertaintiesregardinghowintracellularagentsconnecttoprimarysignals.

第11頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversityOsteoclastdevelopmentanddifferentiation第12頁/共49頁Osteoclastformationandactivity.ShowncentrallyisaschemeofosteoclastdifferentiationinresponsetoM-CSFandRANKL.Abovetheproposeddifferentiationpathwayareexamplesofdirectinhibitors,whilstbelowareindirectinhibitorssignallingthroughintermediatecelltypes.InstituteofMetabolism&Endocrinology,CentralSouthUniversity第13頁/共49頁破骨細胞分化后的主要調(diào)節(jié)通路第14頁/共49頁Cell-to-cellinteractionsinbone

Boneremodelingisadynamicprocess:

requirescoordinatedcellularactivitiesamongosteoblasts,osteocytes,andosteoclastsAdherensjunctions：cadherinsinosteogenesisCommunicatingjunctions：Gapjunctions

Connexin43，45，46，theporeisdifferentDevelopmentandmaintenanceofadifferentiatedosteoblastphenotype.activationoftheERKsignalingcascade

propagationofcalciumwaveInstituteofMetabolism&Endocrinology,CentralSouthUniversity第15頁/共49頁Thecadherinadhesioncomplex.InstituteofMetabolism&Endocrinology,CentralSouthUniversity第16頁/共49頁Asingleconnexinmonomeriscomposedof4transmembranespanningdomains(TM1–4),twoextracellularloops(EC1,2),andacytoplasmicloop(CL).Aconnexon,orhemichannel,iscomposedofahexamericarrayofconnexins.Whendockedwithaconnexononanappositionalcellmembrane,agapjunctionalchannelisformedwhichpermitsthetransferofsmallmoleculessuchasnucleotides,metabolites,andsecondmessengersamongcoupledcells.第17頁/共49頁Cell–cellinteractionsplayanimportantroleinin.uencingandcoordinatingcellfunction.Bothadherensandcommunicativejunctionscanregulatetheabundanceormovementofsignalingmoleculesortranscriptionallyactivefactors,whosecanonicalpathwaysconvergeuponthenucleustomodulategeneexpressionandultimatelycellularfunction.第18頁/共49頁各種細胞間的通訊與偶聯(lián)InstituteofMetabolism&Endocrinology,CentralSouthUniversityChemicalsynapses，gapjunctions,Plasmodesmata,Tunnelingnanotubes第19頁/共49頁Modelofdeathligand/deathreceptorandmitochondria-mediatedclassicalapoptosissignaling,includingBcl-2members.InstituteofMetabolism&Endocrinology,CentralSouthUniversityCell

Apoptosis第20頁/共49頁

Osteoblast/osteocyteapoptosis

apoptoticosteoblastswithtypicalapoptoticfeaturesofcondensedchromatinand/ornuclearfragmentationareseenrarelyinvivo(TUNEL+)theymustbejuxtaposedtoosteoidandtootherosteoblastsbecauseboneformationiscarriedoutbyteamsofosteoblasts,butnotinosteocytes

theyshouldhaveacuboidalmorphologytodistinguishthemfromnearbymarrowcellsandliningcells;specifctechniques,suchasTUNELorISELstainingInstituteofMetabolism&Endocrinology,CentralSouthUniversity第21頁/共49頁第22頁/共49頁OsteoclastApoptosisInducedby:

Bisphosphonatesandestrogenetal.

Phenotype:cellshrinking,chromatincondensation,nuclearfragmentation,andstrongTRAPstainingInhibitby:Srcandintegrinsin,PU.1,c-Fos,NF-kBp50/p52,andNFATc1InstituteofMetabolism&Endocrinology,CentralSouthUniversity第23頁/共49頁Summaryofstimulatoryandinhibitoryfactorsinvolvedinosteoclastdevelopmentandapoptosis.

第24頁/共49頁M-CSF,RANKL,TNF,andIL-1inducedsignalsleadingtoactivationofSrc,Akt,Erks,andmTOR/S6Kpathwaysinosteoclaststoblockapoptosis.InstituteofMetabolism&Endocrinology,CentralSouthUniversity第25頁/共49頁成骨與破骨之間的藕聯(lián)OPG/RANK/RANKLTraidIRSfamilyOthers（membraneadapterDNAX-activatingprotein12/Fcreceptorcommonγchain）第26頁/共49頁RANKLisadendriticcellsurvivalfactor,butalsostronglyinducesosteoclastogenesisandboneresorptionviabindingtoRANK.OPGinhibitsosteolysisandblocksRANKL/RANKinteraction.AlthoughtheOPG/RANK/RANKLtriadisthemainmodulatorofboneapposition/resorptioncoupling,othercontrolsofosteoclasticdifferentiationalsoexist,suchasTNF-aandIL-1,whichareabletomodulatethebiologicalactivitiesofthetriad.第27頁/共49頁TheinitialstepinRANKsignalingisthebindingoftheTRAF6adaptatorproteinwithinthecytoplasmicdomainofRANK.TRAF6thenactsasapivotaladaptorleadingtospecificgeneexpressionregulatingosteoclastdifferentiationandactivation.ThedownstreamtargetsofTRAF6includetranscriptionfactorssuchasNF-kB,aswellasmoleculesinvolvedinsignaltransduction.第28頁/共49頁第29頁/共49頁CentralcontrolofboneremodelingInstituteofMetabolism&Endocrinology,CentralSouthUniversity第30頁/共49頁骨組織細胞功能研究方法InstituteofMetabolism&Endocrinology,CentralSouthUniversity第31頁/共49頁骨組織細胞生物學行為分析InstituteofMetabolism&Endocrinology,CentralSouthUniversityMTT

Growthcurve

Cellcycle

形成礦化結節(jié)的能力

第32頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversity第33頁/共49頁骨組織細胞的聯(lián)合三維培養(yǎng)裝置InstituteofMetabolism&Endocrinology,CentralSouthUniversity破骨細胞培養(yǎng)在骨片上形成骨吸收陷窩第34頁/共49頁Combinedfluorescentandbright-fieldconfocalimagesillustratingpositivestainingforosteocytehypoxiaandactin.Innormalintactbone(A),minimalosteocytehypoxiawasdetected(1.1

±

0.5%).After24

hofdisuse(B),osteocytehypoxiawassignificantlyelevated(8.4

±

1.8%).Ahighlevelofpositiveactinstainingwasevident(C;72

±

9%).InstituteofMetabolism&Endocrinology,CentralSouthUniversity第35頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversity基質(zhì)干細胞的多向誘導性第36頁/共49頁骨質(zhì)疏松的其他分子機制osteoporosisislikelytobecausedbycomplexinteractionsamonglocalandsystemicregulatorsofbonecellfunction.Theheterogeneityofosteoporosismaybeduenotonlytodifferencesintheproductionofsystemicandlocalregulators,butalsotochangesinreceptors,signaltransductionmechanisms,nucleartranscriptionfactors,andenzymesthatproduceorinactivatelocalregulators.Withinthelastdecade,theidentificationofmanyoftheregulatorymechanismsthathavebeenlinkedtoosteoporosishasbeentheresultofgeneticstudies.第37頁/共49頁骨質(zhì)疏松的其他分子機制CentralroleofestrogenCalcium,vitaminD,andparathyroidhormoneReceptoractivatorofNF-κB,itsligand,andosteoprotegerinGenesdeterminingosteoblastdifferentiationandfunctionCytokines,prostaglandins,NO,andleukotrienesCollagenabnormalitiesLeptinandneuralpathways第38頁/共49頁分子生物學研究基礎及方法InstituteofMetabolism&Endocrinology,CentralSouthUniversity報告基因研究方法熒光示蹤第39頁/共49頁基因功能研究的其他手段轉(zhuǎn)基因與基因剔除體外轉(zhuǎn)錄與翻譯交互作用蛋白人工突變調(diào)控區(qū)研究InstituteofMetabolism&Endocrinology,CentralSouthUniversity第40頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversityMMP-1OPGBeta-ActinActinNorthernblotanalysisthechangeofMMPmRNAexpressioninosteoblasttreatedbyestrogenCVitcE2用Western分析雌激素與Vitc對成骨細胞OPG表達的影響Phospho-ERK1/2Total-ERK1/2Control16122448961,25D3

(h)VitD3處理不同時間對細胞周期相關基因磷酸化的影響第41頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversity轉(zhuǎn)錄水平高通量分析方法用含8600個基因的芯片分析SEDT-PA患者成骨細胞基因表達譜的變化用含890個基因的細胞因子表達Array分析雌激素處理前后成骨細胞細胞因子表達譜的變化第42頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversity第43頁/共49頁InstituteofMetabolism&Endocrinology,CentralSouthUniversityTheeffectofcbfa-1inthethedevelopmentofbones;alcianblue(forcartilage);alizarinred(forbone).

A)Wild-typelittermate.(B)Homozygousmutantshowingcartilage,butanabsenceofossificationthroughouttheentirebody.ThetranslocationofP53inducedbyTNFisrepairedbyestrogendetectedbyIF第44頁/共49頁Implicati