PTX阻斷溶血磷脂酸對大鼠胚胎神經(jīng)干細胞的促增殖作用-英文-

PTX阻斷溶血磷脂酸對大鼠胚胎神經(jīng)干細胞的促增殖作用_英文_()文章編號:1007-6611200801-0011-04PTX阻斷溶血磷脂酸對大鼠胚胎神經(jīng)干細胞的促增殖作用123123()崔慧林,喬健天山西醫(yī)科大學組胚教研室,太原030001;山西醫(yī)科大學生理教研室;通訊作者()()摘要:目的觀察溶血磷脂酸lysophosphatidicacid,LPA對大鼠胚胎神經(jīng)干細胞neuralstemcells,NSCs的增殖的影響以()及百日咳毒素pertussistoxin,PTX對LPA作用的影響。方法體外培養(yǎng)神經(jīng)干細胞,對生成的神經(jīng)干細胞球進行計數(shù)顯μ()示增殖情況。結(jié)果?在特殊的無血清培養(yǎng)液中加入低濃度LPA0.01-1.0mol/L后,神經(jīng)干細胞球數(shù)量呈劑量依賴性增加,表明LPA對NSCs有顯著的促增殖作用;?培養(yǎng)液中同時加入LPA和PTX后,神經(jīng)干細胞球計數(shù)顯示LPA引起的NSCs的增殖被抑制了98%。結(jié)論PTX阻斷了LPA對大鼠胚胎神經(jīng)干細胞的促增殖作用,從而推測LPA的促增殖作用可能主要通過PTX敏感的Gi2Ras2Raf2MAPK信號轉(zhuǎn)導途徑實現(xiàn)。關(guān)鍵詞:溶血磷脂酸;神經(jīng)干細胞;增殖;百日咳毒素;大鼠中圖分類號:Q254;Q189文獻標識碼:APTXblocksthepromotiveactionofLPAonproliferationofembryonicneuralstemcellsinrats1231(CUIHui2lin,QIAOJian2tianDeptofHistologyandEmbryology,ShanxiMedicalUniversity,Taiyuan030001,China;23)DeptofPhysiology,ShanxiMedicalUniversity;Correspondingauthor()Abstract:ObjectiveTostudytheeffectoflysophosphatidicacidLPAontheproliferationofratembryonicneuralstemcells()()NSCsinvitroandtheeffectofpertussistoxinPTXonLPA2inducedpromotiveproliferationofNSCs.MethodsNSCswereμ()culturedinvitro,andneurosphereswerecounted.Results?LowerconcentrationsofLPA0.0121.0mol/Ldose2dependent2lyincreasedthenumbersofneurospheresculturedinspecificserum2freemedia,whichindicatedasignificantpromotiveactionofLPAμontheproliferationofNSCs.?AfterNSCsweretreatedby1.0mol/LLPAcombinedwithPTX,98%oftheLPA2inducedprolif2erationwasblocked.ConclusionTheseresultssuggestthatthepromotiveeffectLPAmightbebroughtaboutmainlybythePTX2sensitiveGi2Ras2Raf2MAPKpathwayratherthanthePTX2insensitiveones.Keywords:lysophosphatidicacid;neuralstemcell;proliferation;pertussistoxin;ratsreceptorsweremarkedlyexpressedbyNSCsandLPA3IntroductionwithLPreceptorbeingexpressedfaintlybythe()2A2areoperationallydeNeuralstemcellsNSCs5finedasmitoticallycompetent,self2renewing,andsameNSCs,itisinterestingforustoobservethemultipotentcellsabletodifferentiatealongthreemainpossibleeffectofLPAontheproliferationofembry2(cellarelineagesintheearlyneurogenesisi.e,neu2onicNSCsofratsinvitroandanalyzethesignal1,2)rons,astrocytes,andoligodendrocytes.Sincetransductionpathwaysaswell.theidentificationofNSCs,theiruniquepropertieshavemadeNSCsanattractivesubjectfortherapeuticMaterialsandmethodsapplicationstothedamagedbrain.TheelucidationofMaterialsexternalsignalsthatmightbeinvolvedintheregula2(LysophosphatidicacidLPA,12acyl2sn2glycerol232tionofNSCsinrespectoftheirproliferationanddif2)phosphate,fattyacid2freebovineserumalbuminferentiationmaydevelopareplenishablesourceofre2()()FAFBSA,pertussistoxinPTX,andpoly2L2ly2()placementcells.LysophosphatidicacidLPAisasinewerepurchasedfromSigma.DMEM/F12wassimplephospholipidsmediatingdiversecellularre2fromHyclone.B27wasfromGibco2BRL.Basicfi2sponses,includingcellproliferation,throughtrans2()broblastgrowthfactorbFGFwasfromPepro3,4membraneandintracellularsignalingpathways.Tech.Mousemonoclonalanti2nestinwasfromBDAswehavedemonstratedinratsrecentlythatLPA1Bioscience.SABC2cy3kitwasfromBioster.LPA()基金項目:山西省青年科技基金資助項目20031062()()at15-20DIV,therewerenoattachingcellsandwasdissolvedinphosphate2bufferedsalinePBScontaining4%FAFBSAandstoredasstocksolutiondeadcells,butneurosphereswithphase2brightsus2()1mmol/Lat-20?.pendingrowthmedium.Byimmunocytochemicalex2PrimarycultureandidentificationofNSCsaminationat20DIV,thecellsandtheneurosphereswerestronglynestin2positive,identifyingthecellsareE14ratembryoswereobtainedfromtimed2pregnantWistarratswiththemorningofvaginalplugdesig2NSCsinnature.Thepurityofnestin2positivecellsat()20DIVwas96%.natedasembryonicday0E0.Cellculturesandi2dentificationwereproducedessentiallyasdescribedHowever,asshowninFig1byneurospheres5,6previously.counting,whendifferentconcentrationsofLPA(μ)0.01-10mol/LorvehicleonlywereaddedintoNeurospheres2countingthegrowthmediaat20DIV,respectively,itturnedAsaNSCgeneratesaneurosphere,countingtheoutthatdifferentconcentrationsofLPAexhibitedanumbersofneurospherescanbeusedtoassessthebi2directionalordualactionsontheproliferationofproliferationofNSCs.Neurospheresat20DIVwereNSCs.Thelowerconcentrationslessthan1.0dissociatedbymechanicalaction,thenthedissociatedcellswereplatedinuncoated242wellplatesintheμmol/Lledtoadose2dependentincreaseof2neurogrowthmediumcontainingdifferentconcentrationsofμanin2spheres,itpeakedat1.0mol/Lshowing(μ)creaseofthenumbersofneurospheresbyupto1.69LPA0.01-10mol/Lorvehicleonly.After7d,()thenumbersofneurosphereswerecounted.Toassess?0.15foldP<0.01ascomparedto79?3pertheeffectsofPTX,aninhibitorofGiproteinactiva2wellinthevehiclecontrol;whilehigherconcentra27tion,ontheLPA2inducedproliferationofNSCs,μtionsmorethan1.0mol/Lwereused,theycaused()NSCswerecotreatedwithPTX100ng/mlandlowerandlowerproliferationofcells;andmoreover,μ1.0mol/LLPAorvehicle,respectively,andneuro2whenconcentrationsreachedandsurpassedthelevelspherescountingweremadeatthesametimepoint.μof3-4mol/L,LPAevencauseddegenerationofThenumbersofneurosphereswerecountedunderin2NSCsascomparedtothatincontrols.Thus,1.0()vertedmicroscopeOlympus1M.Theexperimentμbethemosteffectivedosageformol/LLPAmightwasrepeatedfor3timesand6sampleswerecountedpromotingtheproliferationofNSCsinvitroandwasfromeachofthem.usedinthefollowingobservations.StatisticalanalysisThedatawereexpressedaspercentagesofthecontrol()valuesMean?S.E.andweresubmittedtoaone2wayANOVAfollowedbyDunnet’spost2hoctest.DifferencewereconsideredstatisticallysignificantatP<0.05.ResultsEffectsofLPAonproliferationofNSCsAfterculturingthecortexoftheE14ratembryoingrowthmediumfor24h,small2sizedneurospheresFig1EffectsofLPAontheproliferationoftheratem2madebyseveralcellswereobserved,withmanydeadbryonicNSCs.EachdatarepresentedMean?S.Ecellsandcelldebris.At3-4DIV,thenumbersofofsixculturesfromthreeindependentexperimentsneurospheresincreasedandthesizeofthembecame3()P<0.05vsvehiclecontrollargerforcomprisingmorecells.AftertwopassagesAnalysisofsignalingpathwaysofLPAinpromot2NSCs.Inthepresentstudy,wealsoanalyzethepos2siblesignalingpathwaysbywhichLPAbringsaboutingNSCsproliferationitspromotiveactiononproliferationofNSCs.BasedFig2showsthatcotreatmentwithPTXshowednoontheexperimentscarriedoutmainlyonfibroblasts,effectincontrolgroup,butitalmostcompletely34MoolenaarandvanLeeuwenetalhavereported2inducedproliferationofNSCsbyre2blockedtheLPAthatatleastfourGprotein2coupledsignalingpath2ductionrateof98%ascomparedtothatinthecon2wayshavebeenidentifiedfortheactionofLPA:two()trolgrouptreatedwithLPAonlyP<0.01.()ofthemarePTX2sensitivethroughGiproteinand(theothertwoarePTX2insensitivethroughGqpro2)tein.Inourpresentstudy,PTXhasblockedupmostoftheLPA2inducedproliferationofNSCs,andthusexcludesthepossibilitythatPTX2insensitivesig2nalingpathwaysmightplayaratherimportantroleintheblockingprocessestoavisibleextent.Ontheoth2erhand,itisgenerallyacceptedthatoutoftwoPTX2sensitivepathways,oneistoinhibitthemembrane3effectoradenylylcyclaseandtheotheristoactivateRasproteinandthenthedownstreamRaf/MAPKFig2EffectsofPTXontheLPA2inducedproliferationof9cascadetostimulatethecellproliferation.Indeed,3()ratembryonicNSCsP<0.05vsLPAalonesomereportshowedthatLPAdidinduceadecreaseofcAMPthroughGiproteinandthentheinhibitionofDiscussion10adenylylcyclase.However,asindicatedrecentlyOurresultshaveshownthatthelowerconcentrations11byKranenburgandMoolenaar,accordingtotheofLPAcanpromotetheproliferationofNSCs,withdataobtainedmainlyfromtheproliferationofquies2μtheconcentrationof1.0mol/Lbeingthebestforcentfibroblasts,Rasactivationcouldbefullyinhibit2thiseffect;andwhenthedosageincreasescontinu2edbyPTX,yetnotsecondarytoGi2mediatedinhibi2μouslyandsurpassesupto3.0mol/L,LPAexhibitstionofadenylylcyclase.Thus,itisreasonabletodegenerativeactionupontheculturedNSCsascom2proposethatthepromotiveactionofLPAonprolifer2paredtotheblankcontrol,andevencausestotalationofNSCsmightbealmostcompletelybroughtμdeathofcellsonce10mol/LormoreLPAhavebeenaboutthroughtheGi2Ras2Raf/MAPKsignalingpath2reached.Theseresultswereinconsistentwiththatway,leavingafewroomfortheparticipationofGi2obtainedinourpreviousstudiesontheeffectsofLPAAC2cAMPcascadeandotherpathwaysintothepro2upontheproliferationofratembryonicneuralstemmotiveprocesses.Thequestionsthatneedtoanswer36cellsbyH2thymidineincorporation.Contosetremain:westilldonotknowwhatarethemembrane8alhasreportedthatco2incubationwith100nmol/LeffectorandthesecondmessengerintheplausibleGi2ofLPAincreasestheproliferationby25%inculturedRas2Raf/MAPKsignalingpathwayconcerningtheneuroblaststakenfromE12-13mouseembryonicLPA2inducedcellproliferation,andwhyGiproteincorticesfollowing18hexposuretoLPA.SoLPAcan(activateshereratherthan“inhibits”asitgenerallybethoughtasafavouritecandidateforpromotingthe)doestheRasanddownstreamsubstratesinthesig2proliferationofNSCs.nalingpathwaythatweareinterestedin.These5OurrecentreportshowedthatLPandLPA1A3problemsawaitfurtherinvestigation.Insummary,receptorsweremarkedlyexpressedbyNSCswithourresultshaveshownthatLPAatitslowerconcen2LPreceptorbeingexpressedfaintlybythesameA2trationsstronglypromotestheproliferationofNSCsrentiationtocholinergicneuronsinvitroJ.ShengliXuebao,byactivatingtheGi2Ras2Raf2MAPKsignalingpath22006,58:547-555.waytostimulatethemitogenicactivityinthesecellsvanCorvenEJ,HordijkPL,MedemaRH,etal.Pertussistoxin27throughLPandLPreceptors.A1A3sensitiveactivationofp21rasbyGprotein2coupledreceptorReferencesagonistsinfibroblastsJ.ProcNatlAcadSciUSA,1993,90:12SeabergRM,vanderKooyD.Stemandprogenitorcells:thepre1257-1261.maturedesertionofrigorousdefinitionsJ.TrendsNeurosci,ContosJJ,FukushimaN,WeinerJA,etal.Requirementforthe82003,26:125-131.lpA1lysophosphatidicacidreceptorgeneinnormalsucklingbe22GageFH,RayJ,FisherLJ.Isolation,characterization,anduseofhaviorJ.ProcNatlAcadSciUSA,2000,97:13384-13389.stemcellsfromtheCNSJ.AnnuRevNeurosci,1995,18:1599AnS,BleuT,ZhengY,etal.RecombinanthumanGprotein2-192.coupledlysophosphatidicacidreceptorsmediateintracellularcal23MoolenaarWH.Lysophosphatidicacid,amultifunctionalphos2ciummobilizationJ.MolPharmacol,1998,54:881-888.pholipidmessengerJ.JBiolChem,1995,270:12949-12952.