腺相关病毒介导PSA基因转染DC的实验研究

腺相关病毒介导PSA基因转染DC的实验研究

尤长宣罗荣城梁卫江

南方医科大学南方医院肿瘤中心TheOncologyDepartmentofNanfangHospital,theSouthernMedicalUniversity,GuangzhouAdaptiveandInnateImmuneSystemsSkin&MucousMembranesrapidlyregeneratingsurfaces,peristalticmovement,mucociliaryescalator,vomiting,flowofurine/tears,coughingCellularandHumoralDefenseslysozyme,sebaceous/mucoussecretions,stomachacid,commensalorganisms,complementproteins,phagocytosis,naturalkillercellsInvasion&infectionBarriersInnateimmunityAdaptiveimmunityInflammationCellularandHumoralDefensesAntibodies,cytokines,helperTcells,cytotoxicTcells〔CTL〕MK-0069-01Ag/MHCClassIIAg/MHCClassICD4ActivationCytokinesCD8TARGETLYSIS

Activation〔CTL〕Dendriticcell-MHCClassIrestricted-antigenspecificAntigenpresentationTumorantigenAntigenpositivetumorcellDendriticcellsexpressavarietyofcellsurfacemoleculeswhichplaykeyrolesintheirinteractionswithTcells.TheMHCmoleculespresentantigenicfragments(peptides)toTcellsinaformthatcanberecognizedinahighlyspecificfashionbytheappropriateTcellreceptor,whichisuniqueforeveryTcellandeveryantigen.Thecostimulatorymolecules(CD80andCD86)providea"second-signal"thatamplifiestheantigenrecognitionprocess.AccessorymoleculeslikeICAMsandLFAsenhancethephysicalinteractionbetweenTcellsandDCs.DendriticCellProperties

前列腺癌细胞大量表达PSAPSA是前列腺细胞内的一种特异性蛋白；——前列腺癌标志物．选择PSA作为前列腺癌目标抗原．Loadingdendriticcellswith

theantigen１．Protein/peptideloading２．GenedeliveryloadingDisadvantagesofProtein/peptideloadingT1/2ofproteins/peptidesisveryshort.Allarerapidlylost.2)MostprotocolsforgeneratingcytotoxicTlymphocytes〔CTL〕take3-5weeks.GenedeliveryloadingThegenecanproduceaconstantandlargeamountofprotein〔ＰＳＡ〕!!!!!VectorsforGenedeliveryRetroviruses(Retrov)(8kb,RNA,enveloped)

Adenoviruses(Ad)(35kb,DNA,non-enveloped)

Adeno-AssociatedVirus(AAV)(5kb,DNA,non-enveloped)

Retroviruses(Retrov)(8kb,RNA,enveloped)1)TransgeneinactivationbyDNAmethylation．2)Causescancer: proviralchromosomalintegrationactivatesproto-oncogene．3)Retrovirusvectorsareeasilydestroyed,easilyinactivated.Adenoviruses(Ad)(35kb,DNA,non-enveloped)-arehighlyimmunogenic,causinginflammationandcelldeath，thatisthenon-therapeuticimmuneresponse-cannotinfectnon-dividingcells〔Ｇ０〕Adeno-AssociatedVirus(AAV)

(5kb,DNA,non-enveloped)

non-pathogenic

lowimmuneresponsechromosomalintegration

longtermexpressioninvivo

infectdividingandnon-dividingcells

Ag/MHCClassIIAg/MHCClassICD4ActivationTarget：PSACytokinesCD8TARGETLYSIS

Activation(CTL)DendriticCell-MHCClassIRestricted-AntigenSpecificOverviewofImmuneFunctionsrAAV/

PSA

ProtocolofDendriticCellsGeneration.Harvestnon-adherentCellsNonadherentCells+IL-2Incubate2-4hr,37oC

AdherentCellsLymphocytes0.5x107/3mlperwellBloodFicollIsopaqueDiluteplasma

lymphocytesRedcellsgranulocytesplateletsrAAVinfection

Chromiumreleaseassayprimingday0123456789101112131415GM-CSFIL-4IL-2andIL-7MixedwithT-cellsorPBLTNF-Structureofexperimentsday3day5day7CTLDay551Cr释放法检测CTL对靶细胞杀伤的抗原特异性51Cr释放法检测CTL对靶细