摘要
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MHC-Ⅱ、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MHC-Ⅱ、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 explore response of tumor-specific cytotoxic T lymphocyte（ CTL） induced by dendritic cells（ DCs） sensitized with exosomes that derived from heat-shocked gastric cancer cell. Methods： exosomes from mice murine foregastric cancer（ MFC） cells（ Exo）,heat-shocked MFC cells（ Exo / HS）,and lysates（ Lys） of MFC cells were prepared,respectively. Morphology of the exosomes was observed by electron microscopy and protein components of the exosomes were detected by Western blotting. DCs generated from murine bone marrow were cultured and sensitiaed with Exo /HS,Exo and Lys,and the prepared DCs vaccines were termed as DC-Exo / HS,DC-Exo,and DC-Lys,respectively. Also,DCs were sensitized with exosomes from heat-shocked MFC tumor cells that transfected with HSP70 siRNA. Phenotypes of DCs were detected by flow cytometry. Mice were immunized with DC-Exo / HS,DC-Exo,DC-Ly,DC,or PBS respectively; the proliferation of splenic T cells was measured by3H-TdR and activity of spleen CTL was measured by LDH.MFC cells-bearing mouse models were constructed to examine immunotherapy effects of the DCs vaccines. Results： exosomes were confirmed as small membranous vesicles with electron microscopy. Detection of Western blotting showed that Exo / HS contained high level of HSP70. Flow cytometry demonstrated that the exosomes from heat-shocked MFC cells could significantly up-regulate expressions of MHC-Ⅱ,CD80,CD86 and CD40 molecules on the DCs,and interference with HSP70 siRNA could down-regulate expressions of MHC-Ⅱ,CD80,CD86 and CD40 molecules on the DCs sensitized with the exosomes from heat-shocked MFC tumor cells. Results of3H-TdR showed that proliferation ability of T cells in DC-Exo / HS group was markedly enhanced as compared with those in DC-Exo,DC-Lys,DC and PBS groups（ P〈0. 01）.Results of LDH showed that higher level of CTL activity was induced in DC-Exo / HS group as compared with those in DCExo,DC-Lys,DC or PBS（ P〈0. 01）,and induced CTL activity in HSP70 siRNA group was sig
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2016年第2期188-194,共7页
Chinese Journal of Cancer Biotherapy
基金
浙江省自然科学基金资助项目(No.LY14HY160011)~~
