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目的：探讨热激胃癌细胞来源外泌体致敏树突状细胞（dendritic cells, DCs）诱导的肿瘤特异性细胞毒T细胞（cytotoxic T lymphocyte, CTL）反应。方法：制备小鼠前胃癌细胞（ murine foregastric cancer cells，MFC）来源外泌体（Exo）、热激MFC细胞来源外泌体（Exo/HS）及MFC细胞冻融抗原（lysates, Lys），通过电镜观察外泌体的形态，Western blotting检测外泌体的成分。培养小鼠骨髓来源的DCs，将Exo/HS、Exo和Lys冲击致敏DCs，制备的DCs瘤苗分别命名为DC-Exo/HS、DC-Exo和DC-Lys。HSP70小干扰RNA（small interference RNA, siRNA）转染MFC细胞，热激后分离上清，用制备外泌体致敏DCs，流式术检测DCs的表型。以DC-Exo/HS、DC-Exo、DC-Lys、DC和PBS免疫小鼠，3H-TdR法检测脾脏T细胞的增殖，LDH法检测脾细胞CTL活性。建立MFC细胞荷瘤小鼠模型，观察各组DCs瘤苗的免疫治疗效应。结果：电镜确认外泌体为膜性小囊泡。Western blotting检测表明，Exo/HS含有高水平的HSP70，流式术发现热激MFC细胞来源外泌体能显著上调DCs表面MHCⅡ、CD80、CD86 和CD40分子的表达，HSP70 siRNA干扰能下调热激MFC细胞来源外泌体刺激的DCs表面 MHCⅡ、CD80、CD86和CD40分子的表达。3H-TdR检测结果显示DC-Exo/HS刺激T细胞增殖的能力显著强于DC-Exo、DC-Lys、DC和PBS组（均P<0.01），LDH检测结果表明DC-Exo/HS诱导的CTL活性显著高于DC-Exo、DC-Lys、DC和PBS组（均P<0.01），HSP70 siRNA组诱导的CTL活性显著低于对照siRNA组（P<0.01）。免疫治疗结果显示，DC-Exo/HS对荷瘤小鼠的肿瘤抑制效应显著优于其他各组（P<0.01）。结论：热激胃癌细胞来源外泌体致敏的DCs能诱导显著的抗肿瘤免疫反应。
Objective: To investigate the tumor-specific cytotoxic T lymphocyte (CTL) responses induced by dendritic cells (DCs) derived from exosome-secreting cells of heat-shock gastric cancer cells. Methods: Preparation of Mouse gastric carcinoma cells (murine foregastric cancer cells, MFC) source exosomes (Exo), heat shock MFC cell-derived exosomes (Exo / HS) and MFC cell lysates (lysates, Lys), by The morphology of exosomes was observed by electron microscope and the composition of exosomes was detected by Western blotting. DCs derived from mouse bone marrow were cultured, and DCs were primed by Exo / HS, Exo and Lys shocks. The DCs vaccine were named DC-Exo / HS, DC-Exo and DC-Lys respectively. HSP70 siRNA was transfected into MFC cells, and the supernatant was isolated after heat shock. DCs were primed with exosomes and flow cytometry was used to detect the phenotype of DCs. The mice were immunized with DC-Exo / HS, DC-Exo, DC-Lys, DC and PBS. The proliferation of splenic T cells was detected by 3H-TdR method. The establishment of MFC cell tumor-bearing mouse model to observe the immunotherapy effect of DCs vaccine in each group. Results: Electron microscopy confirmed that exosomes were membranous vesicles. Western blotting analysis showed that, Exo / HS contain high levels of HSP70, heat shock flow cytometry found that cell-derived exosomes MFC regeneration DCs significantly upregulated surface MHCⅡ, CD80, CD86 and CD40 molecules, HSP70 siRNA interference can be reduced thermal Expression of MHC Ⅱ, CD80, CD86 and CD40 on the surface of DCs stimulated by MFC cell-derived exosomes. 3H-TdR test results showed that DC-Exo / HS ability to stimulate T-cell proliferation was significantly stronger than DC-Exo, DC-Lys, DC and PBS group (P & lt; 0.01), LDH test results show that DC-Exo / HS-induced CTL activity was significantly higher than that of DC-Exo, DC-Lys, DC and PBS groups (all P <0.01). The CTL activity induced by HSP70 siRNA group was significantly lower than that of control siRNA group (P <0.01). Immunotherapy results showed that DC-Exo / HS on tumor-bearing mice tumor inhibitory effect was significantly better than the other groups (P <0.01). CONCLUSION: DCs sensitized with exosomes derived from heat shock gastric cancer cells can induce significant antitumor immune response.