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1、腺相关病毒介导腺相关病毒介导PSAPSA基因基因转染转染DCDC的实验研究的实验研究尤长宣 罗荣城 梁卫江南方医科大学南方医院肿瘤中心The Oncology Department of Nanfang Hospital,the Southern Medical University,Guangzhou Adaptive and Innate Immune Systems Skin&Mucous Membranesrapidly regenerating surfaces,peristaltic movement,mucociliary escalator,vomiting,flow of u
2、rine/tears,coughingCellular and Humoral Defenseslysozyme,sebaceous/mucous secretions,stomach acid,commensal organisms,complement proteins,phagocytosis,natural killer cellsInvasion&infectionBarriersInnate immunityAdaptive immunityInflammationCellular and Humoral DefensesAntibodies,cytokines,helper T
3、cells,cytotoxic T cells（CTL）MK-0069-01Ag/MHC Class IIAg/MHC Class IIAg/MHC Class IAg/MHC Class IActivationActivationCytokinesCytokinesTARGET LYSIS ActivationActivation（CTL）Dendritic cell-MHC Class I-MHC Class I restricted restricted-antigen-antigen specific specificAntigen presentationTumor antigenA
4、ntigen positive tumor cellDendritic cells express a variety of cell surface molecules which play key roles in their interactions with T cells.The MHC molecules present antigenic fragments(peptides)to T cells in a form that can be recognized in a highly specific fashion by the appropriate T cell rece
5、ptor,which is unique for every T cell and every antigen.The costimulatory molecules(CD80 and CD86)provide a second-signal that amplifies the antigen recognition process.Accessory molecules like ICAMs and LFAs enhance the physical interaction between T cells and DCs.Dendritic Cell Properties 前列腺癌细胞大量
6、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Loading dendritic cells with the antigenProtein/peptide loadingGene delivery loading Disadvantages of Protein/peptide loading1)T1/2 of proteins/peptides is very short.All are rapidly lost.2)Most protocols for generating cytotoxic T lymphocytes（CTL）take 3
7、-5 weeks.Gene delivery loading The gene can produce a constant and large amount of protein（）（）!Vectors for Gene deliveryRetroviruses(Retrov)(8kb,RNA,enveloped)Adenoviruses(Ad)(35kb,DNA,non-enveloped)Adeno-Associated Virus(AAV)(5kb,DNA,non-enveloped)Retroviruses(Retrov)(8kb,RNA,enveloped)1)Transgene
8、inactivation by DNA methylation2)Causes cancer:proviral chromosomal integration activates proto-oncogene 3)Retrovirus vectors are easily destroyed,easily inactivated.Adenoviruses(Ad)(35kb,DNA,non-enveloped)-are highly immunogenic,causing inflammation and cell death，that is the non-therapeutic immune
9、 response-can not infect non-dividing cells（）Adeno-Associated Virus(AAV)(5kb,DNA,non-enveloped)non-pathogenic low immune response chromosomal integration long term expression in vivo infect dividing and non-dividing cells Ag/MHC Class IIAg/MHC Class IIAg/MHC Class IAg/MHC Class IActivationActivation
10、Target Target：PSAPSACytokinesCytokinesTARGET LYSISTARGET LYSIS ActivationActivation(CTL(CTL)Dendritic CellDendritic Cell-MHC Class IMHC Class I Restricted Restricted-Antigen-Antigen Specific SpecificOverview of Immune FunctionsrAAV/PSA Protocol of Dendritic Cells Generation.Harvestnon-adherentCellsN
11、on adherentCells+IL-2Incubate2-4hr,37oC AdherentCellsLymphocytes 0.5x107/3ml per wellBloodFicollIsopaqueDilute plasma lymphocytesRed cells granulocytesplateletsrAAV infection Chromium release assayprimingday 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15GM-CSFIL-4IL-2 and IL-7Mixed with T-cells or PBLTNF-Structure of experimentsday 3day5day 7CTL Day 551Cr释放法检测释放法检测CTL对靶细胞杀伤的抗原特异性对靶细胞杀伤的抗原特异性51Cr释放法检测释放法检测CTL对靶细胞杀伤的对靶细胞杀伤的MHC I 类限制性类限制性 研究结论研究结论成功构建了AAV/PSA质粒，其能够高效转染DC；AAV/PSA基因成功整合入DC染色体内；DC内成功表达PSA蛋白；AAV/PSA转染DC后，仅1周即可诱导得到大量、高活性的CTL；所获得CTL对PSA阳性的前列腺癌细胞具有特异、高效的细胞毒杀伤效应。Thank you!Thank you!