【作者】 席泓；

【导师】 张学光； 顾宗江；

【作者基本信息】 苏州大学 ， 免疫学， 2002， 硕士

【摘要】 机体的抗肿瘤免疫主要为T淋巴细胞所介导，T细胞的致敏、激活、扩增和对肿瘤细胞的杀伤作用均有赖于抗原递呈细胞肿瘤递呈相应的抗原肽及相关因子的参与。树突状细胞（DC）是体内功能最强的专职抗原递呈细胞，在机体免疫应答中起极其重要的作用。为了进一步研究DC在激发和诱导抗原特异性细胞毒性T细胞（CTL）中的作用及其机制，寻找以DC为基础的CTL体外有效扩增方案，我们采用外周血单个核细胞（PBMCs）来源的DC，负载由激发型CD40mAb联合⁶⁰Coγ辐照诱导B淋巴瘤细胞株Daudi的凋亡，成熟的肿瘤抗原肽-DC进一步激发和诱导自体的T细胞，联合激发型CD28mAb、IL-2、IL-15、IL-7等不同因子体外扩增肿瘤特异性CTL，在此基础上建立优化的体外扩增方案；ELISA检测所扩增T细胞上清中细胞因子IL-12、IL-10、IFN-γ的产生；³H-TdR掺入试验和⁵¹Cr释放试验分别测定CTL的增殖和细胞毒效应。结果显示（1）凋亡肿瘤细胞负载联合CD40mAb激发可使DC上调表达CDla、CD80、CD86和HLA-DR，并能促进IL-12的分泌；（2）凋亡肿瘤细胞负载后的DC在激发型CD40mAb作用下，能激发和扩增出高效、特异杀伤靶细胞的CTL；（3）肿瘤抗原负载DC联合激发型CD28mAb、IL-15、IL-2诱导扩增肿瘤特异性CTL效率最高。结论：CD40mAb激发的凋亡肿瘤细胞负载 DC在 CD28mAb、ILJ 和 IL上的联合作用下可有效激活和扩增肿瘤特异性CTh。更多还原

【Abstract】 Dendritic cells (DC), the initiator and modulator of immune response , are the most powerful professional antigen-presenting cells (APC). Recent studies indicated that DC have the most power to activate tumor specific CTL, therefore, DC are applied in the therapy of tumors, e. g: B cell lymphoma, prostate cancer, multiple myeloma, breast cancer. But the optimal method for induction of tumor specific CTL hasn’t been determined.Our studies focused on the role and mechanism of DC in the activation and proliferation of tumor antigen specific CTL and establishing an optimal method for producing tumor antigen specific CTL in vitro. We used PBMC-derived DC loaded with apoptotic Daudi cells, which were irradiated by 60Co Y after incubation with agonist CD40mAb ( 5cl 1, 5 ? g/ml) for 24h. Tumor specific CTL were activated by the mature apoptotic-tumor-cell-loaded DC in the presence of IL-2 and CD28mAb, IL-15, IL-7. The expression of membrane molecules on DC and CTL were analyzed by FCM; IL-12, IL-10, IFN- Y concentrations of T cell culture supernatants were measured by ELISA; Proliferation andcytotoxicity of CTL were analyzed by 3H-thymidine incorporation and a 4-h 51Cr release assay respectively. Our results showed that (1) following incubation with agonist CD40mAb (5 ?g/ml) ,then irradiated by 60Co ? , the apoptotic rate of Daudi cells was significantly increased; (2) After treatment of CD40mAb (5 ?g/ml), the expressions of CD la, CD80, CD83, CD86 , HLA-DR on tumor-loaded DC were upregulated and concentration of IL-12 in the supernatant were increased. (3) Apoptotic-tumor-cell-loaded DC, in addition with agonist CD28mAb, IL-2, IL-15 could induce efficient quality and large quantity of tumor specific cytotoxic T lymphocytes, it also could downregulate the expression of Fas on CTL. Conclusion: Apoptotic-tumor-cell-loaded DC induced maturation by CD40mAb could efficiently induce the activation and proliferation of tumor specific CTL, and the combination of IL-2, IL-15, CD28mAb was a very powerful method to expand antigen specific CTL in vitro.更多还原