代谢性骨病分子、细胞生物学.ppt

周后德2005 4 20长沙 InstituteofMetabolism Endocrinology CentralSouthUniversity 代谢性骨病细胞 分子生物学基础及研究方法与策略 代谢性骨病 代谢性骨病是一类伴有钙 磷 维生素D代谢失调以及甲状旁腺功能 细胞因子与激素分泌异常的全身性骨骼病 代谢性骨病的分类 1 骨质疏松2 佝偻 软骨病3 原发性甲状旁腺机能亢进症4 原发性甲状旁腺机能减退症5 中毒性骨病 维生素D 氟 镉 铅 磷等中毒 6 其他如粘多糖代谢异常 Marfan症 Paget症 SEDT PA osteoporosis Representsacontinuum inwhichmultiplepathogeneticmechanismsconvergetocauselossofbonemassandmicroarchitecturaldeteriorationofskeletalstructure Thesefactors coupledwithanincreasedriskoffalls contributetoahighincidenceoffragilityfracturesinosteoporoticpatients Skeletalfragilitycanresultfrom a failuretoproduceaskeletonofoptimalmassandstrengthduringgrowth b excessiveboneresorptionresultingindecreasedbonemassandmicroarchitecturaldeteriorationoftheskeleton c aninadequateformationresponsetoincreasedresorptionduringboneremodeling Theboneremodelingunitsorbonemulticellularunits BMUs 骨组织的细胞成份 成骨谱系 间充质干细胞 骨源性始组细胞 osteoprogenitorcells 成骨细胞 osteoblast 骨衬细胞 bone liningcell 骨细胞 osteocyte 破骨谱系 多潜能造血干细胞 破骨细胞 osteoclast 成软骨细胞 软骨细胞 破软骨细胞 InstituteofMetabolism Endocrinology CentralSouthUniversity 非细胞成分 胶原非胶原蛋白骨矿物质血管神经 InstituteofMetabolism Endocrinology CentralSouthUniversity 组织细胞学基础 InstituteofMetabolism Endocrinology CentralSouthUniversity Osteoblastdevelopmentanddifferentiation Transcriptionfactors Surfacemarkers EffectsofWntsignalingonosseouscells ThecanonicalWntsignalingpathwaypromotestheproliferation expansionandsurvivalofpre andimmatureosteoblasts Dkks Sfrps andWif 1antagonizeWntsignalinginosteoblaststofacilitatedeathofimmaturecells buttheymayalsodownregulatethepathwayinmaturecellstoinduceterminaldifferentiation Wntsignalinginosteoblastsandbonediseases Osteoclastdevelopmentanddifferentiation Normaldifferentiationrequires 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 InstituteofMetabolism Endocrinology CentralSouthUniversity Osteoclastdevelopmentanddifferentiation Osteoclastformationandactivity ShowncentrallyisaschemeofosteoclastdifferentiationinresponsetoM CSFandRANKL Abovetheproposeddifferentiationpathwayareexamplesofdirectinhibitors whilstbelowareindirectinhibitorssignallingthroughintermediatecelltypes InstituteofMetabolism Endocrinology CentralSouthUniversity 破骨细胞分化后的主要调节通路 Cell to cellinteractionsinbone Boneremodelingisadynamicprocess requirescoordinatedcellularactivitiesamongosteoblasts osteocytes andosteoclastsAdherensjunctions cadherinsinosteogenesisCommunicatingjunctions GapjunctionsConnexin43 45 46 theporeisdifferentDevelopmentandmaintenanceofadifferentiatedosteoblastphenotype activationoftheERKsignalingcascadepropagationofcalciumwave InstituteofMetabolism Endocrinology CentralSouthUniversity Thecadherinadhesioncomplex InstituteofMetabolism Endocrinology CentralSouthUniversity Asingleconnexinmonomeriscomposedof4transmembranespanningdomains TM1 4 twoextracellularloops EC1 2 andacytoplasmicloop CL Aconnexon orhemichannel iscomposedofahexamericarrayofconnexins Whendockedwithaconnexononanappositionalcellmembrane agapjunctionalchannelisformedwhichpermitsthetransferofsmallmoleculessuchasnucleotides metabolites andsecondmessengersamongcoupledcells Cell cellinteractionsplayanimportantroleinin uencingandcoordinatingcellfunction Bothadherensandcommunicativejunctionscanregulatetheabundanceormovementofsignalingmoleculesortranscriptionallyactivefactors whosecanonicalpathwaysconvergeuponthenucleustomodulategeneexpressionandultimatelycellularfunction 各种细胞间的通讯与偶联 InstituteofMetabolism Endocrinology CentralSouthUniversity Chemicalsynapses gapjunctions Plasmodesmata Tunnelingnanotubes Modelofdeathligand deathreceptorandmitochondria mediatedclassicalapoptosissignaling includingBcl 2members InstituteofMetabolism Endocrinology CentralSouthUniversity CellApoptosis Osteoblast osteocyteapoptosis apoptoticosteoblastswithtypicalapoptoticfeaturesofcondensedchromatinand ornuclearfragmentationareseenrarelyinvivo TUNEL theymustbejuxtaposedtoosteoidandtootherosteoblastsbecauseboneformationiscarriedoutbyteamsofosteoblasts butnotinosteocytestheyshouldhaveacuboidalmorphologytodistinguishthemfromnearbymarrowcellsandliningcells specifctechniques suchasTUNELorISELstaining InstituteofMetabolism Endocrinology CentralSouthUniversity OsteoclastApoptosis Inducedby Bisphosphonatesandestrogenetal Phenotype cellshrinking chromatincondensation nuclearfragmentation andstrongTRAPstainingInhibitby Srcandintegrinsin PU 1 c Fos NF kBp50 p52 andNFATc1 InstituteofMetabolism Endocrinology CentralSouthUniversity Summaryofstimulatoryandinhibitoryfactorsinvolvedinosteoclastdevelopmentandapoptosis M CSF RANKL TNF andIL 1inducedsignalsleadingtoactivationofSrc Akt Erks andmTOR S6Kpathwaysinosteoclaststoblockapoptosis InstituteofMetabolism Endocrinology CentralSouthUniversity 成骨与破骨之间的藕联 OPG RANK RANKLTraidIRSfamilyOthers membraneadapterDNAX activatingprotein12 Fcreceptorcommon chain RANKLisadendriticcellsurvivalfactor butalsostronglyinducesosteoclastogenesisandboneresorptionviabindingtoRANK OPGinhibitsosteolysisandblocksRANKL RANKinteraction AlthoughtheOPG RANK RANKLtriadisthemainmodulatorofboneapposition resorptioncoupling othercontrolsofosteoclasticdifferentiationalsoexist suchasTNF aandIL 1 whichareabletomodulatethebiologicalactivitiesofthetriad TheinitialstepinRANKsignalingisthebindingoftheTRAF6adaptatorproteinwithinthecytoplasmicdomainofRANK TRAF6thenactsasapivotaladaptorleadingtospecificgeneexpressionregulatingosteoclastdifferentiationandactivation ThedownstreamtargetsofTRAF6includetranscriptionfactorssuchasNF kB aswellasmoleculesinvolvedinsignaltransduction Centralcontrolofboneremodeling InstituteofMetabolism Endocrinology CentralSouthUniversity 骨组织细胞功能研究方法 InstituteofMetabolism Endocrinology CentralSouthUniversity 骨组织细胞生物学行为分析 InstituteofMetabolism Endocrinology CentralSouthUniversity MTT Growthcurve Cellcycle 形成矿化结节的能力 InstituteofMetabolism Endocrinology CentralSouthUniversity 骨组织细胞的联合三维培养装置 InstituteofMetabolism Endocrinology CentralSouthUniversity 破骨细胞培养在骨片上形成骨吸收陷窝 InstituteofMetabolism Endocrinology CentralSouthUniversity InstituteofMetabolism Endocrinology CentralSouthUniversity 基质干细胞的多向诱导性 骨质疏松的其他分子机制 osteoporosisislikelytobecausedbycomplexinteractionsamonglocalandsystemicregulatorsofbonecellfunction Theheterogeneityofosteoporosismaybeduenotonlytodifferencesintheproductionofsystemicandlocalregulators butalsotochangesinreceptors signaltransductionmechanisms nucleartranscriptionfactors andenzymesthatproduceorinactivatelocalregulators Withinthelastdecade theidentificationofmanyoftheregulatorymechanismsthathavebeenlinkedtoosteoporosishasbeentheresultofgeneticstudies 骨质疏松的其他分子机制 CentralroleofestrogenCalcium vitaminD andparathyroidhormoneReceptoractivatorofNF B itsligand andosteoprotegerinGenesdeterminingosteoblastdifferentiationandfunctionCytokines prostaglandins NO andleukotrienesCollagenabnormalitiesLeptinandneuralpathways 分子生物学研究基础及方法 InstituteofMetabolism Endocrinology CentralSouthUniversity 报告基因研究方法 荧光示踪 基因功能研究的其他手段 转基因与基因剔除体外转录与翻译交互作用蛋白人工突变调控区研究 InstituteofMetabolism Endocrinology CentralSouthUniversity InstituteofMetabolism Endocrinology CentralSouthUniversity MMP 1OPG Beta ActinActin NorthernblotanalysisthechangeofMMPmRNAexpressioninosteoblasttreatedbyestrogen CVitcE2 用Western分析雌激素与Vitc对成骨细胞OPG表达的影响 Phospho ERK1 2 Total ERK1 2 Control1612244896 1 25D3 h VitD3处理不同时间对细胞周期相关基因磷酸化的影响 InstituteofMetabolism Endocrinology CentralSouthUniversity 转录水平高通量分析方法 用含8600个基因的芯片分析SEDT PA患者成骨细胞基因表达谱的变化 用含890个基因的细胞因子表达Array分析雌激素处理前后成骨细胞细胞因子表达谱的变化 InstituteofMetabolism Endocrinology CentralSouthUniversity InstituteofMetabolism Endocrinology CentralSouthUniversity Theeffectofcbfa 1inthethedevelopmentofbones alcianblue forcartilage alizarinred forbone A Wild typelittermate B Homozygousmutantshowingcartilage butanabsenceofossificationthroughouttheentirebody ThetranslocationofP53inducedbyTNFisrepairedbyestrogendetectedbyIF Implicationforthediagnosisandtreatmentofosteoporosis Pharmacotherapyfo