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 The Oncology Department of Nanfang Hospital, the Southern Medical University, Guangzhou,Adaptive and Innate Immune Systems,Skin & Mucous Membranes rapidly regenerating surfaces, peristaltic movement, mucociliary escalator, vomiting, flow of urine/tears, coughing,Cellular and Humoral Defenses lysozyme, sebaceous/mucous secretions, stomach acid, commensal organisms, complement proteins, phagocytosis, natural killer cells,Invasion & infection,Barriers Innate immunity Adaptive immunity,Inflammation,Cellular and Humoral Defenses Antibodies, cytokines, helper T cells, cytotoxic T cells（CTL）,MK-0069-01,Ag /MHC Class II,Ag/ MHC Class I,CD4,Activation,Cytokines,CD8,TARGET LYSIS,Activation （CTL）,Dendritic cell,- MHC Class I restricted - antigen specific,Antigen presentation,Tumor antigen,Antigen positive tumor cell,Dendritic 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 receptor, 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,Loading dendritic cells with the antigen,Protein/peptide loading,Gene delivery loading,Disadvantages of Protein/peptide loading T1/2 of proteins/peptides is very short. All are rapidly lost. 2) Most protocols for generating cytotoxic T lymphocytes（CTL） take 3-5 weeks.,Gene delivery loading,The gene can produce a constant and large amount of protein（）!,Vectors for Gene delivery Retroviruses (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 inactivation by DNA methylation 2) 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 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 II,Ag/ MHC Class I,CD4,Activation,Target ：PSA,Cytokines,CD8,TARGET LYSIS,Activation(CTL),Dendritic Cell,- MHC Class I Restricted - Antigen Specific,Overview of Immune Functions,rAAV/ PSA,Protocol of Dendritic Cells Generation,.,Harvest non-adherent Cells,Non adherent Cells + IL-2,Incubate 2-4hr, 37oC,Adherent Cells,Lymphocytes 0.5x107/3ml per well,Blood,Ficoll Isopaque,Dilute plasma,lymphocytes,Red cells granulocytes platelets,rAAV infection,Chromium release assay,priming,day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15,GM-CSF,IL-4,IL-2 and IL-7,Mixed with T-cells or PBL,TNF-,Structure of experiments,day 3 day5 day 7,CTL Day 5,51Cr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MHC I 类限制性,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!,
