热休克E.G7-OVA肿瘤细胞来源的exosomes的抗肿瘤作用  

Enhanced antitumor effects of exosomes derived from heat-shocked E.G7-OVA tumor cells

作　　者：钟海均[1] 杨云山[1] 马胜林[1] 毛伟敏[1] 张沂平[1] 修方明[2] 蔡志坚[2] 陈玮琳[2] 王青青[2] 

机构地区：[1]浙江省肿瘤医院化疗中心,杭州310022 [2]浙江大学免疫学研究所

出　　处：《中华微生物学和免疫学杂志》2010年第2期164-168,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省医药卫生科研基金（2008B023）

摘　　要：目的 本研究探讨热休克E.G7-OVA肿瘤细胞来源的exosomes的体内抗肿瘤效应.方法 通过分级离心和蔗糖密度梯度离心法分离和纯化E.G7-OVA肿瘤细胞来源的exosomes.热休克E.G7-OVA肿瘤细胞来源的exosomes和未热休克E.G7-OVA肿瘤细胞来源的exosomes分别命名为Exo/HS和Exo.通过电镜观察exosomes的形态,Western blot检测exosomes的蛋白成分.以Exo、Exo/HS、PBS免疫小鼠,用E.G7-OVA肿瘤细胞进行攻击,观察各组免疫保护效应;建立E.G7-OVA荷瘤小鼠模型,观察各组免疫治疗效应.通过LDH法检测各组免疫小鼠脾细胞CTL活性.结果 电镜下exosomes为双层膜的囊泡样结构,直径为40～100 nm.Western blot结果 表明：Exo和Exo/HS都含有HSC70、HSP70、HSP60、HSP90、MHC Ⅰ和OVA分子,而Exo/HS中HSP70和MHC Ⅰ分子的含量更高.免疫保护试验发现,Exo/HS组免疫小鼠90 d的无瘤率显著高于Exo组和PBS组（50%、20%、0%,P〈0.01）;对荷瘤小鼠的免疫治疗显示,Exo/HS对荷瘤小鼠的肿瘤抑制效应显著高于Exo组和PBS组（P〈0.01）.CTL结果 表明,Exo/HS免疫小鼠诱导的OVA抗原特异性的CTL活性显著高于Exo组和PBS组（P〈0.01）.结论 热休克E.G7-OVA肿瘤细胞来源的exosomes可作为有效的肿瘤疫苗.Objective To study the antitumor effects of exosomes derived from heat-shocked E.G7-OVA tumor cells in vivo. Methods Exosomes derived from E.G7-OVA tumor cells were isolated and purified by serial centrifugation and sucrose gradients ultracentrifugation. Exosomes from heat-shocked or non-heat-shocked E.G7-OVA tumor cells were named as Exo/HS and Exo correspondingly. Exosomes were viewed by electron microscopy. Protein components of exosomes were detected by Western blot. Exo, Exo/ HS or PBS were injected into mice before injection of E.G7-OVA tumor cells, and antitumor effects were ob-served in each group. Mouse model bearing E.G7-OVA tumor cells were established to examine immunother-apy effects of Exo or Exo/HS. Cytotoxity of spleen CTL were measured by LDH. Results Exosomes con-tained bi-layer membrane and their diameters are between 40 nm and 100 nm under electron microscopy. The Western blot results showed that HSC70, HSP70, HSP60, HSP90, MHC Ⅰ and OVA were present in both Exo and Exo/HS. However, Exo/HS contained more HSP70 and MHC Ⅰ than Exo. Protective antitu-mor immunity suggested that tumor-free survival （90 days） rate in Exo/HS vaccinated mice was significantly higher than those in Exo or PBS vaccinated mice （50%, 20%, 0%, P〈0.01）. Therapeutic antitumor effects showed that immunization by Exo/HS resulted in dramatically enhanced antitumor effects when com-pared to the Exo- or PBS-treated groups （P〈0.01）. CTL results showed that immunization with Exo/HS in-duced higher level of OVA-specific CTL responses as compared with those from Exo or PBS （P〈0.01）. Conclusion Exosomes derived heat-shocked E.G7-OVA tumor cells may be used as potent cancer vaccine.

关 键 词：热休克 E.G7-OVA细胞 EXOSOMES 抗肿瘤效应 

分 类 号：R730.2[医药卫生—肿瘤]