Exosomes联合卡介苗的体内抗肿瘤效应

In vivo anti-tumor effect of tumor cell-derived exosomes combined with BCG

目的:探讨exosomes(Exo)联合卡介苗(bacillus Calmette-Gurin vaccine,BCG)的体内抗肿瘤效应。方法:通过密度梯度离心法分离和纯化E.G7-OVA肿瘤细胞来源的Exo,Western blotting检测其蛋白成分。分别以Exo、BCG、Exo+BCG、PBS免疫小鼠,以E.G7-OVA细胞攻击,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。LDH法检测4组免疫小鼠脾细胞CTL细胞毒性。结果:Western blotting检测显示,Exo含有HSP60、OVA、HSC70和CD63分子;免疫保护实验结果显示,Exo+BCG组免疫小鼠90d无瘤率显著高于Exo组和BCG组(60%vs20%、0%,P<0.01);免疫治疗实验结果显示,Exo+BCG对小鼠移植瘤的抑制显著强于Exo组和BCG组(P<0.01)。CTL检测结果显示,Exo+BCG免疫小鼠的CTL细胞特异性杀伤E.G7-OVA细胞的活性显著高于其他各组(P<0.01)。结论:BCG作为免疫佐剂能显著增强exosomes的体内抗肿瘤效应。

Objective： To study the in vivo anti-tumor effect of exosomes （Exo） combined with bacillus CalmetteGuerin vaccine（BCG）. Methods： Exo was isolated and purified from culture supernatant of E. G7-OVA tumor cells by density gradient centrifugation. Protein components of Exo were detected by Western blotting. Exo, BCG, Exo combined with BCG （ Exo ＋ BCG） or PBS were pre-injected into mice before injection of E. G7-OVA cells, and the anti-tumor effects were observed in each group. Mouse model bearing E. G7-OVA cells was established to examine the immuno-therapy effects of Exo with or without BCG. Cytotoxity of spleen CTL was measured by LDH in different groups. Results ： Exo derived from E. G7-OVA cells contained HSP60, OVA, HSC70 and CD63 as detected by Western blotting. Tumor-free rate at 90 d was significantly higher in Exo ＋ BCG vaccinated mice than those in Exo or BCG vaccinated mice as measured by immuno-protective assay （60% vs 20% or 0% ,P 〈 0.01 ）. Immuno-therapy assay showed that tumor inhibitory effect in Exo ＋ BCG group was significantly higher than those in Exo or BCG groups （P 〈 0.01 ）. CTL results showed that CTL of Exo ＋ BCG vaccinated mice had significantly enhanced ability to specifically kill target E. G7-OVA cells compared with those of Exo and BCG groups （P 〈 0.01 ）. Conclusion： BCG as an immuno-adjuvant can significantly enhance the antitumor effect of exosomes in vivo.