树突细胞分泌的exosomes生物学特性及功能的初步研究

A preliminary study on the biological characteristics and function of exosomes derived from dendritic cells

目的建立树突细胞（DC）分泌的exosomes的制备方法，分析其生物学特性及其在抗肿瘤免疫中的功能。方法从正常人外周血单个核细胞中诱导未成熟DC（imDC），并使其负载K562细胞的抗原，然后用脂多糖（LPS）诱导DC成熟（mDC），通过超速离心结合膜超滤的方法分别提取imDC和mDC分泌的exosomes。检测exosomes的粒径并通过电子显微镜分析exosomes的形态，Western blot法检测exosomes的表面分子。比较mDC和imDC分泌的exosome8引起的针对K562抗原特异性T细胞的增殖、CD69的上调、细胞因子IFN．1的分泌及对肿瘤细胞的杀伤能力。结果制备的exosomes为碟状小囊泡，平均直径为72．3nm，表达CD80、CD86、HLA．DR、FasL、CD54和乳脂肪球-表皮生长因子-因子Ⅷ（MFG-E8）。与imDC相比，mDC分泌的exosomes CD80表达较高而MFG-E8的表达较低，并且在体外mDC分泌的exosomes能够更显著地引起特异性T细胞的增殖和免疫应答：在exosomes最适浓度下，T细胞增殖的吸光度值为O．50±0．01，激活T细胞的CD69上调，并且有（13．4±5．8）％T细胞扩增，（22．8±2．4）％的T细胞产生细胞因子IFN-1，对肿瘤细胞的杀伤率为（21．3±8．6）％。结论建立了简便、快速的exosomes分离纯化方法。

Objective To establish a method for isolating exosomes from dendritic cells （DC）, and to analyse its biological characteristics and function in antitumor immunity. Methods Immature DCs （im- DC） from human peripheral blood mononuclear cells were loaded with the antigen of K562 tumor cells, then exosomes were secreted from imDC and lipepelysaccharide （LPS） induced mature DC （mDC）. The exosomes from iml）C and mDC were isolated separately by ultracentrifugation and ultrafiltration. The exosomes diameter was determined, their profile was observed by electron microscope, and the surface molecules were detected by Western blot. To analyse the effect of exosemes on antitumor immunity, the proliferation, IFN-γ, expression, CI）69 up-regulation and cytotoxicity of antigen-specific T cells were measured. Results Exosomes were small flattened sphere vesicles with an average diameter of 72.3 nm and expressed CD80, CD86, HLA-DR, FasL, CD54 and MFG-E8 molecules. As compared to immature exosomes, exosomes from mDC were proved to express more CDS0 and less MFG-E8, to be more potent for inducing antigen-specific T cells proliferation and immunity respond in vitro： at its optimum concentration, the absorption value of T cell proliferation test was 0.50 ± 0. 01, CD69 was up-regulated and （ 13.4 ± 5.8） % of T cells was in proliferating, （ 22.8 ± 2.4） % of T cells expressed IFN-γ, and （21.3 ± 8.6） % of tumor cells were killed. Conclusion A simple and quick method to isolate and analyse exosomes is established. The exosomes can induce antitumor immunity respond.