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[doc] Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic cell on mice of H22 liver cancer

By Barbara Peterson,2014-09-08 02:22
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[doc] Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic cell on mice of H22 liver cancer

    Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic

    cell on mice of H22 liver cancer

obiliaryDepartment,FirstHospital‟

    ;MicrobiologyDepartment,Medicalcollege‟

    ;Xi‟anJiaotongUniversity,Xi‟an710061,China

    ;Xi‟anJiaotongUniversity.Xi‟an710061,China

    ;lAbstract]Objective:ToimproveDCbasedtumorvaccination,westudied

    whetherdendriticceils

    ;(DCs)whichcoculturedwithH22livercancercellsderivedheatshockprot

    ein(HSP)glycoprotein96

    ;(gP96)affecttheTcellactivatingpotentialinvitroandtheinductionoftumorimmunityinvivo?Meth?

    ;ods:MaturationofmurinebonemarrowderivedDCwasinducedbyGM

    CSFplusIL4,whichmimiced

    ;theimmunostimulatoryeffectofDC.CocuhuredDCandgp96

    peptidecomplexeswereusedtovaccineH22

    ;livercancerce11sofmice.Usingmurinemodelswecomparedtheimmunogenecityof19t2modifiedbygp96?

    ;peptidescomplexesderivedfrommurinelivercancercellsaloneorinactivetu

morcells.Toverifythespeci

    ;ficitvofthevaccine,iitroassayswereexecuted.Serumcytokinelevelswerequantifiedtoexplorethe

    ;supposedpathwayofDCmodifiedbygp96peptidecomplexesanditseffectonantitumorimmuneresponse?

    ;ResuIts:DCmodifiedbygp96

    peptidecomplexescanactivatespleenlymphocyteandthelattercanspecifi

    ;callvkillH22cellsbutnotEhrilichascitescarcinomacells.ModifiedDCcanin

    antigen ducepotenttumor

    ;speeifieimmuneresponse,augmenttheproliferationofThlcells,andinhibittumorgrowth.Conclusion:

    ;Inthisstudy,wehavedevelopedanovelDCmediatedtumorvaccinebycom

    bingthegp96antigenicpep

    ;tidescomplexesandinducingimmuneresponseagainstspecifictumorcells.gp96canbeidentifiedasapo

    ;tentDCactivator.

    ;[Keywords]dendriticcell;gp96peptidecomplexes;1ivercancer;vaccine

    ;Increasingevidencehasestablishedthatimmu

    ;nizationwithheatshockprotein(HSP)isolated

    ;fromcancercellsorvirusinfectedcellscanelicit

    ;protectiveimmuneresponse.Thisparadigmhas

    ;alsobeensubstantiatedbyHSPglycoprotein96

;(gp96)whichcanimmunizeandinduceimmunere

    ;sponseagainstspecificantigens[„.Dendriticcells

    ;(DCs)arehighlyspecializedantigenpresentingcells ;(APC)thatareuniqueintheirpotencytoinduceT ;celldependentAgspecificimmuneresponses4].A

    ;numberofinvestigatorshavedemonstratedthatDC

    ;basedvaccinescangeneratespecificantitumorim

    ;munityinmurinetumormodelsc一川.Thesevaccines

    ;werebasedonfusionorcocuhureofDCandtumor ;cellsontumorpeptideortumorlysatepulsed

    ;DCc.,.BecauseHSPhasthepromiscuousabilityto ;

    ;:SupportedbytheNationalNaturalScienceFoundationof ;China(No.30200369)

    ;

    ;:Correspondingauthor.Email:yangwei@21cn.com

    ;chaperoneandpresentabroadrepertoireoftumor ;antigenstoAPC[..wereasonedthattheymayim

    ;provethetherapeuticpotentialofDC——basedvac——

    ;cines.Towardthisgoal,westudiedwhethermodi

    ;fiedDCswhichcocuhuredwithH221ivercancer ;cellsderivedgp96affectstheTcellactivatingpo-

    ;tentialinvitroandtheinductionoftumorimmunity ;invivo.

    ;MATERIALSANDMETHoDS

    ;MiceBALB/cmice,aged6-8weeksand

    20gwereprovidedbyLaboratorialAn—— ;weighted18——

    ;imalsCenterofFourthMilitaryMedicalUniversity ;andmaintainedunderpathogenfreeconditions.An

    ;imalcarewasprovidedinaccordancewiththeproce

    ;duresoutlinedinthe”ModernMedicalLaboratory

    ;Animals”(People‟sMilitaryMedicalPress,1999).

    ;CelllinesH22livercancercellsandEhrilich ;ascitescarcinomacellsweregiftsofTeachingand ;ResearchingRoomofImmunologyandPathology, ;

    ;980rofMedicalCollegesofPLA2006;21(2) ;Xi‟anJiaotongUniversityandmaintainedinRP—

    ;MI1640supplementedwith10%fetalbovine ;SerUii1-

    ;Isolationofgp96peptidecomplexesgp96was

    ;purifiedfromH22livercancercellofBALB/c ;mouseasdescribedc?.Inbrief,carcinomatous ;asciteswaschallengedwithasecondheattreatment

;at42Cfor12hinwater,homogenizedin30

;mmol/LNaHC03,O.5mmol/LPMSF(pH7.1),

    ;byrepeatedtwiceoffreezingandthawingcycles.A ;100000gsupernatantwasobtainedandappliedto ;ConAsepharoseaffinitychromatographycolumn ;(AmershamPharmaciaBiotech)whichequilibrated ;with30mmol/LNaHC03.Theglycoproteinswere ;elutedby1Oamethylmannoside.Theeluatewas

    ;appliedtoMonoQFPLCcolumn(AmershamPhar

    ;maciaBiotech),equilibratedwith20mmol/LTris ;?

    ;HCl(DH8.5),0.5mol/LNaClandelutedovera ;20600mmol/LNaC1gradient.Thesamplebuffer ;ateverypeakswerecollectedandfolloweddialyzing ;andconcentrating.PreparationsanalyzedbySDS

    ;PAGEsilverstainingandWesternblotwithanti

    ;Grp94antibody(StressGensCo).

    ;PreparationofDCinvitroBALB/cmicebone ;marrowwasflushedfromfemursandtibiaswith ;physiologicalsaline.Afterlysedredbloodcellsby ;hyposmsis,theremainingswerewashed3times ;withHank‟ssolution.Themurinebonemarrowde

;rivedprecursercellswereplated10./Lto6-wellcul

    ;tureplatesandmaintainedinRPMI1640culture ;mediumwith10FCSinthepresenceof100ng/ml

    CSFand10ng/mlrecombi ;recombinantmouseGM

    ;nantmouseIL4(AmershamPharmaciaBiotech). ;1ooselyadherentcellswereharvestedonday3and ;day7toverifybyS100immunohistochemistryand ;electronmicroscopicphotograph.Finally,the10./L ;DCscoculturedwith250ngpurifiedgp96-peptide ;complexesat37C,5%Co2for2h.

    ;ImmunizationFortyBALB/cmicewereallo

    ;catedto4groups(10).Forconstructingmurine

    ;livercancermodels,10./LH22livercancercells ;weregivensubcutaneouslyattherootofrightback ;leg.After3doftumorchallenge,fortherapeutic ;immunization,2×10./LmodifiedDC,6ug/ml ;gp96proteininactivatedH22livercancercells,for ;hlankcontrol,thephysiologicalsaline(blankcon

    ;trolgroup)wereinjectedtomiceevery3d,totally ;4timesaltogether.

    ;ELISAandC,releaseassayInoculatedmice ;werekilledafter21d.Centrifugateofmouseserum

;werecollectedandthelevelsofIL10andIFN-7

    ;weredeterminedbyELISAkit(EndogenCo.).The ;cytotoxicitywasevaluatedbyastandardC(pro

    ;videdbyPathologicalTeachingandResearching ;RoomofXi‟anJiaotongUniversity)releaseassay.

    ;H22cellcollectedfromfreshhydropertoneumwere ;usedasatarget(at1000cellsperwel1)ina6-hour ;assay.Toassesstumorspecific,theEhrilichascites ;carcinomacellsfromfreshhydropertoneumwereal

    ;sousedasatarget.Theproportionofeffectspleen ;cellsandtargetcellswas25:l-TheCI(specific ;killrate)wasevaluatedbyfollowedformula:CI ;(experimentaltremacpmnaturallyreleased

    ;tremacpm)/(maxreleasedtremacpmnaturally

    ;releasedtremacpm)×100.

    ;StatisticalanalysisTheKruskalWallisHtest

    ;wasusedforstatisticalanalysis.

    ;RESULTS

    ;DCculturePlentyo{nonadhereroundcells

    ;wereobservedattheinitialstageofculture.Great ;changeappearedafter3d.Theburringdendritic ;couldbeseenonthecell‟sbodyattheday4(Fig

;1),andthecellsweretypicallysuspended,gradual

    ;ly,thecellsgrewinclusters.After7d,theloosely ;adherentcellswerespreadaroundthecultureflask ;equably.Whenweobservedthroughscanningelec

    ;tronmicroscope(SEM),thebiggercells‟body

    ;couldbeseen.Plentyofbiforkeddendriticplasma ;tubergrewonthemembraneofburringcells.Some ;oftuberpresentedsliceshapeaccordingwiththe ;topicalshapecharacterofDC(Fig2).DCwas ;stainedbyimmunohistochemicalmethodpresenting ;S100proteinstrongpositiveterracotta(Fig3).

    ;ConcentratiOnchangeofIFN-Y.IL-IOin ;serumTheconcentrationofIFNrwasthehighest

    ;inDCimmunitygroup,butthelowestincontrol ;group,andgreatdifferenceexistedamongevery ;group(H26.542,P0.0000).Theconcentra

    ;tionofIL10wasthelowestinDCimmunitygroup, ;

    ;

    ;““,(,3*ledi~(,(„,Ih‟o./Pt1200(~:(2)

    ;Fig1Ckaiigesofdentlritic~elts

    ;A:IKI>11dv1iilrrI‟iI.uilFI1?1il_m-l?IriiI?iit.1I-.Inl:.1:L-l1.‟…l{

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    ;r?(i1hi:llicaf1cr2Idl,fchaI1nl(td1)yH2‟2Iivcr

    ;,iIiC(-r?II.TIiwL1gh1)fSLIhc__iJ]OLI:tlm()rttI ;“ltwl87n.1,…mc,f…1Ut‟g1-OtlI,.

    ;(I:_.1,1gi[1gliprottink~rotllJ.t281

    ;I‟0‟inH22Iiv1.,rf.-~incerLIl14rl?1).ndthe ;(:inlngt(}Ll)is(1i..II.?nr”tarkuhie

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    ;dividualvarience.HSPbasedvaccinesworkacross ;tumortypesandbypasstheneedforidentifyingthe

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