胃保护素对破骨细胞的作用PPT课件

.,1,GhrelinisanOsteoblastMitogenandIncreasesOsteoclasticBoneResorptionInVitro,.,2,Ghrelin(胃动素、胃肠动素)是生长激素促分泌物质受体(growthhormonesecretagoguereceptor,GHS-R)的内源性配体.Ghrelin的结构：Ghrelin是由28个氨基酸组成的小分子多肽，它有两种形式：Ghrelin-28和des-Gln14-ghrelin,除了第14位氨基酸Gln的缺失,des-Gln14-ghrelin生物活性与Ghrelin相同。GhrelinN端第3位丝氨酸残基上的n-辛酰基（n-octanoylgroup）对生物活性具有重要作用。人和大鼠的Ghrelin在进化上高度保守，仅11位和12位上相差两个氨基酸。,BackgroundKnowledge,.,3,Structuresofhumanandratghrelins,Bothhumanandratghrelinsare28-aminoacidpeptides,inwhichSer3ismodifiedbyafattyacid,primarilyn-octanoicacid.Thismodificationisessentialforghrelinsactivity.,K：赖氨酸，A丙氨酸;R:精氨酸，V:缬氨酸,.,4,Thestructureofghrelinreceptor,GHSR,GHSRisaseventransmembrane-domainG-proteincoupledreceptor.TwotranscriptsareproducedfromthehumanGHSRgenebyalternativesplicing:GHSR1aencodesthefunctionalreceptor,andGHSR1b,whichistruncatedaftertransmembranedomain5,appearstobeunabletostimulateintracellularsignalingpathways.,.,5,Signaltransductionpathway,GHsecretagogues(GHSs)stimulateGHreleasethroughtheGHSreceptor(GHS-Rorghrelinreceptor)toincreaseintracellularCa2(Ca2i)levels.GHSsareartificialmoleculesanddonotexistinnature,sothereisnostructuralhomologybetweenghrelinandpeptideGHSs.,.,6,Summary,Ghrelinisreleasedinresponsetofasting,suchthatcirculatinglevelsarehighestimmediatelypriortomeals.Ghrelinincreasedthebone-resorbingactivityofratosteoclasts,butdidnotalterosteoclastdifferentiationinamurinebonemarrowassaynorboneresorptioninexvivocalvarialcultures.,Ghrelinshowedmitogenicactivityinosteoblasts,withastrongeffectinhumancellsandaweakereffectinratosteoblasts.,.,7,1.Introduction,Boneturnoverisacutelyresponsivetofoodintake,withboneresorptionincreasedduringfastingandsuppressedduringfeeding.Numerousfactors,suchasnutritionalcontentorsizeofmeals,mayplayaroleinthisrelationshipandthereisevidencethatsomeguthormones,suchasglucagon-likepeptide-23,4,alsoaffectthenormalpostprandialreductionofboneresorption.,.,8,Anumberofstudiesinvestigatedtheeffectsofghrelininskeletaltissue.AstudyoftheexpressionofGHSR1ainhumantissuesdemonstratedaweakpositivesignalinbonemarrow。Osteoclastresponsetoghrelinhasneverbeeninvestigatedpreviously.Theaimofthecurrentstudywastofurtherinvestigatetheactivityofghrelininbonecells,andinparticularitspossibleroleintheacutestimulationofboneresorptionthatoccursduringfasting.,.,9,AcylatedghrelinBSA(牛血清白蛋白)GibcobrandFBS(胎牛血清)andmediawereusedTritiatedthymidine（氚化胸腺嘧啶）Tartrate-resistantacidphosphatase(TRAP)stainingkit(抗酒石酸酸性磷酸酶染色试剂盒试)1,25-dihydroxyvitaminD3osteoprotegerin(OPG)（骨保护素）salmoncalcitonin（鲑鱼降钙素）,Materials,.,10,Osteoclastdifferentiationinmousebonemarrowcultures,1,25V-D3(10nM),0,2,5d,2,4d,GhrelinC(0.1-10nM),7d,TRAPstaining,MNC,48-wellplates,Mixedmarrowcells,Themixedpopulationofcellsfromthebonemarrowisculturedinthepresenceof1,25-dihydroxyvitaminD3thatpromotesosteoclastformation,.,11,Result,Osteoclastdifferentiationinmousebonemarrowculturesisnotaffectedbyghrelin.,OPG:Recombinanthumanosteoprotegerin(10ng/mL)wasincludedasapositivecontrolforinhibitionofosteoclastogenesis.,.,12,Ghrelinincreasedthebone-resorbingactivityofratosteoclastsinvitro,Bovineboneslices,Matureosteoclast,Incubatefor24h,GhrelinC:1nM/10nM,fixedandstainedforTRAP,TRAP(+),Count,Scrubbing,Reflectedlightimageofresorptionpitsformedbyratosteoclastsonbovineboneslices,Stainanddry,.,13,Results,Ghrelinataconcentrationof1nMinduceda30%increaseinthenumberofpitsexcavatedbyosteoclasts,whereasat10nMtherewasonlya15%increasewhichwasnotsignificantlydifferentfromthecontrol.,Thepotentinhibitorofosteoclastactivity,salmoncalcitonin(1010M),wasusedasapositivecontrol.,.,14,Osteoclastactivityinmousecalvarialcultureisnotaffectedbyghrelin.,Calvariaelabeledinvivowith45Cawerecollectedandincubatedexvivowithexperimentalsubstancesorvehiclefor48hours.,Valuesareexpressedaspercentreleased45Cadetectedovertotal45Cainjected(3Ci).PTH:parathyroidhormone(positivecontrol).,.,15,Ghrelinismitogenictoprimaryhumanosteoblasts.,3H-thymidineincorporationwasmeasuredasanindicatorofcellproliferation.,Humanprimaryosteoblastsincubatedfor24or48hoursinthepresenceof0.1nMghrelinshoweda2-foldincreaseinproliferation,asmeasuredbythymidineincorporation.,Howtodeterminethymidineincorporation?,.,16,Ghrelinweaklyinducedcellproliferationinprimaryratosteoblasts,witha1.1-1.2-foldstimulationofthymidineincorporation(a)andasimilar,modestincreaseincellnumbers(b).,.,17,Conclusion,Ghrelinincreasedthebone-resorbingactivityo