胃保护素对破骨细胞的作用

GhrelinisanOsteoblastMitogenandIncreasesOsteoclasticBoneResorptionInVitro 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 Structuresofhumanandratghrelins Bothhumanandratghrelinsare28 aminoacidpeptides inwhichSer3ismodifiedbyafattyacid primarilyn octanoicacid Thismodificationisessentialforghrelin sactivity K 赖氨酸 A丙氨酸 R 精氨酸 V 缬氨酸 Thestructureofghrelinreceptor GHSR GHSRisaseventransmembrane domainG proteincoupledreceptor TwotranscriptsareproducedfromthehumanGHSRgenebyalternativesplicing GHSR1aencodesthefunctionalreceptor andGHSR1b whichistruncatedaftertransmembranedomain5 appearstobeunabletostimulateintracellularsignalingpathways Signaltransductionpathway GHsecretagogues GHSs stimulateGHreleasethroughtheGHSreceptor GHS Rorghrelinreceptor toincreaseintracellularCa2 Ca2 i levels GHSsareartificialmoleculesanddonotexistinnature sothereisnostructuralhomologybetweenghrelinandpeptideGHSs Summary Ghrelinisreleasedinresponsetofasting suchthatcirculatinglevelsarehighestimmediatelypriortomeals Ghrelinincreasedthebone resorbingactivityofratosteoclasts butdidnotalterosteoclastdifferentiationinamurinebonemarrowassaynorboneresorptioninexvivocalvarialcultures Ghrelinshowedmitogenicactivityinosteoblasts withastrongeffectinhumancellsandaweakereffectinratosteoblasts 1 Introduction Boneturnoverisacutelyresponsivetofoodintake withboneresorptionincreasedduringfastingandsuppressedduringfeeding Numerousfactors suchasnutritionalcontentorsizeofmeals mayplayaroleinthisrelationshipandthereisevidencethatsomeguthormones suchasglucagon likepeptide 2 3 4 alsoaffectthenormalpostprandialreductionofboneresorption Anumberofstudiesinvestigatedtheeffectsofghrelininskeletaltissue AstudyoftheexpressionofGHSR1ainhumantissuesdemonstratedaweakpositivesignalinbonemarrow Osteoclastresponsetoghrelinhasneverbeeninvestigatedpreviously Theaimofthecurrentstudywastofurtherinvestigatetheactivityofghrelininbonecells andinparticularitspossibleroleintheacutestimulationofboneresorptionthatoccursduringfasting 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 Osteoclastdifferentiationinmousebonemarrowcultures 1 25V D3 10nM 0 2 5d 2 4d GhrelinC 0 1 10nM 7d TRAPstaining MNC 48 wellplates Mixedmarrowcells Themixedpopulationofcellsfromthebonemarrowisculturedinthepresenceof1 25 dihydroxyvitaminD3thatpromotesosteoclastformation Result Osteoclastdifferentiationinmousebonemarrowculturesisnotaffectedbyghrelin OPG Recombinanthumanosteoprotegerin 10ng mL wasincludedasapositivecontrolforinhibitionofosteoclastogenesis Ghrelinincreasedthebone resorbingactivityofratosteoclastsinvitro Bovineboneslices Matureosteoclast Incubatefor24h GhrelinC 1nM 10nM fixedandstainedforTRAP TRAP Count Scrubbing Reflectedlightimageofresorptionpitsformedbyratosteoclastsonbovineboneslices Stainanddry Results Ghrelinataconcentrationof1nMinduceda30 increaseinthenumberofpitsexcavatedbyosteoclasts whereasat10nMtherewasonlya15 increasewhichwasnotsignificantlydifferentfromthecontrol Thepotentinhibitorofosteoclastactivity salmoncalcitonin 10 10M wasusedasapositivecontrol Osteoclastactivityinmousecalvarialcultureisnotaffectedbyghrelin Calvariaelabeledinvivowith45Cawerecollectedandincubatedexvivowithexperimentalsubstancesorvehiclefor48hours Valuesareexpressedaspercentreleased45Cadetectedovertotal45Cainjected 3 Ci PTH parathyroidhormone positivecontrol Ghrelinismitogenictoprimaryhumanosteoblasts 3H thymidineincorporationwasmeasuredasanindicatorofcellproliferation Humanprimaryosteoblastsincubatedfor24or48hoursinthepresenceof0 1nMghrelinshoweda2 foldincreaseinproliferation asmeasuredbythymidineincorporation Howtodeterminethymidineincorporation Ghrelinweaklyinducedcellproliferationinprimaryratosteoblasts witha1 1 1 2 foldstimulationofthymidineincorporation a andasimilar modestincreaseincellnumbers b Conclusion Ghrelinincreasedthebone resorbingactivityofra