【摘要】 目的探讨负载肿瘤抗原的树突状细胞(DCs)及其衍生exosomes诱导T细胞活化和特异性肿瘤杀伤的作用。方法将患者静脉血单个核细胞经GM-CSF、IL-4诱导产生DCs,收集培养上清液,超速离心法制备exosomes。流式细胞术检测DCs表面标志表达,用SDS-PAGE和western-blot检测exosomes携带MHC和共刺激分子的情况。MTT法观察DCs及exosomes对T细胞促增殖作用和CTL体外杀伤活性。结果肿瘤抗原致敏的DC高表达MHC-Ⅰ、MHC-Ⅱ、CD54、CD80和CD86,与抗原致敏前相比有显著差异,P<0.05。DC衍生的exosomes表达MHC-Ⅱ、CD54和CD86,并显著引起T细胞扩增和细胞毒效应,与对照组相比有显著差异,P<0.01。结论从脑胶质瘤患者静脉血诱导出高表达MHC和共刺激分子的DCs以及exosomes,具有活化T细胞并产生特异性细胞毒活性的功能,有望成为临床治疗脑胶质瘤有效的新疫苗。更多还原

【Abstract】 Objective To investigate the antitumor efficiency of the special cytotoxic T lymphocytes activated by dendritic cells (DCs) and its exosomes derived from peripheral blood mononuclear cells (PBMCs) of malignant glioma patients. Methods Malignant glioma cells were prepared by primary culture in vitro. PBMCs were cultured with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human interleukin -4 (rhIL-4) to produced DCs. The DCs were collected and their exosomes were separated from culture supernatant by the method of centrifugation (1×10~5g). MHC-Ⅰ, MHC-Ⅱ, and co-stimulatory molecules on the surface of DCs and exosomes were analyzed by FCM, SDS-PAGE, and Western blotting. The mixed lymphocyte reaction (MLR) and cytotoxicity of CTLs to glioma cells were assayed by MTT colorimetry. Results DCs and exosomes pulsed with either apoptotic tumor cells(DC/Apo)or total tumor ribonucleic acid (DC/RNA) or repeated freeze-thawing tumor antigen (DC/Lys) exhibited more MHC-Ⅰ, MHC-Ⅱ, CD54, CD80, and CD86, compared unpulsed DCs (P< 0.05). DC/Apo and DC/RNA induced superior tumor cytolytic activities compared with DC/Lys or controls. DC/Apo induced the greatest expansion. Conclusion DCs and exosomes obtained in vitro from PBMCs and express high levels of MHC-I, MHC-II, and co-stimulatory molecules. The DCs and exosomes present tumor antigen to T cells and enhance the cytotoxicity of CTLs, which may be an effective immunotherapy for intracranial glioma.更多还原