腺相关病毒介导PSA基因转染DC的实验研究.ppt

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,Guangzhou,AdaptiveandInnateImmuneSystems,Skin&MucousMembranesrapidlyregeneratingsurfaces,peristalticmovement,mucociliaryescalator,vomiting,flowofurine/tears,coughing,CellularandHumoralDefenseslysozyme,sebaceous/mucoussecretions,stomachacid,commensalorganisms,complementproteins,phagocytosis,naturalkillercells,Invasion&infection,BarriersInnateimmunityAdaptiveimmunity,Inflammation,CellularandHumoralDefensesAntibodies,cytokines,helperTcells,cytotoxicTcells（CTL）,MK-0069-01,Ag/MHCClassII,Ag/MHCClassI,CD4,Activation,Cytokines,CD8,TARGETLYSIS,Activation（CTL）,Dendriticcell,-MHCClassIrestricted-antigenspecific,Antigenpresentation,Tumorantigen,Antigenpositivetumorcell,DendriticcellsexpressavarietyofcellsurfacemoleculeswhichplaykeyrolesintheirinteractionswithTcells.TheMHCmoleculespresentantigenicfragments(peptides)toTcellsinaformthatcanberecognizedinahighlyspecificfashionbytheappropriateTcellreceptor,whichisuniqueforeveryTcellandeveryantigen.Thecostimulatorymolecules(CD80andCD86)provideasecond-signalthatamplifiestheantigenrecognitionprocess.AccessorymoleculeslikeICAMsandLFAsenhancethephysicalinteractionbetweenTcellsandDCs.,DendriticCellProperties,前列腺癌细胞大量表达PSA,PSA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,Loadingdendriticcellswiththeantigen,Protein/peptideloading,Genedeliveryloading,DisadvantagesofProtein/peptideloadingT1/2ofproteins/peptidesisveryshort.Allarerapidlylost.2)MostprotocolsforgeneratingcytotoxicTlymphocytes（CTL）take3-5weeks.,Genedeliveryloading,Thegenecanproduceaconstantandlargeamountofprotein（）!,VectorsforGenedeliveryRetroviruses(Retrov)(8kb,RNA,enveloped)Adenoviruses(Ad)(35kb,DNA,non-enveloped)Adeno-AssociatedVirus(AAV)(5kb,DNA,non-enveloped),Retroviruses(Retrov)(8kb,RNA,enveloped)1)TransgeneinactivationbyDNAmethylation2)Causescancer:proviralchromosomalintegrationactivatesproto-oncogene3)Retrovirusvectorsareeasilydestroyed,easilyinactivated.,Adenoviruses(Ad)(35kb,DNA,non-enveloped)-arehighlyimmunogenic,causinginflammationandcelldeath，thatisthenon-therapeuticimmuneresponse-cannotinfectnon-dividingcells（）,Adeno-AssociatedVirus(AAV)(5kb,DNA,non-enveloped)non-pathogeniclowimmuneresponsechromosomalintegrationlongtermexpressioninvivoinfectdividingandnon-dividingcells,Ag/MHCClassII,Ag/MHCClassI,CD4,Activation,Target：PSA,Cytokines,CD8,TARGETLYSIS,Activation(CTL),DendriticCell,-MHCClassIRestricted-AntigenSpecific,OverviewofImmuneFunctions,rAAV/PSA,ProtocolofDendriticCellsGeneration,.,Harvestnon-adherentCells,NonadherentCells+IL-2,Incubate2-4hr,37oC,AdherentCells,Lymphocytes0.5x107/3mlperwell,Blood,FicollIsopaque,Diluteplasma,lymphocytes,Redcellsgranulocytesplatelets,rAAVinfection,Chromiumreleaseassay,priming,day0123456789101112131415,GM-CSF,IL-4,IL-2andIL-7,MixedwithT-cellsorPBL,TNF-,Structureofexperiments,day3day5day7,CTLDay5,51Cr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对靶细胞杀伤的MHCI类限制性,研