Exosomes对L1210肿瘤细胞攻击的免疫保护作用研究

Study on immune protection effect of exosomes against attack of L1210 tumor cells

目的：观察L1210小鼠白血病细胞分泌的exosomes的抗肿瘤作用。方法：采用离心超滤和蔗糖密度梯度离心法分离L1210细胞释放的exosomes。固相免疫电镜法制备exosomes的H-2D，ICAM-1电镜标本，免疫印迹观察exosomes中HSP70的表达情况。exosomes免疫DBA／2小鼠，检测特异性细胞毒性T淋巴细胞（CTL）活性；免疫后的小鼠以L1210肿瘤细胞攻击，观察免疫保护效果。结果：L1210细胞分泌的exosomes为直径介于50～100nm的膜性微囊，圆形或椭圆形。抗H-2D，ICAM-1抗体-胶体金免疫电镜标记，囊外膜可见点状、颗粒状电子致密物沉积。通过免疫印迹证明其表达HSP70。Exosomes免疫小鼠，可诱导产生针对L1210细胞的CTL反应；L1210的exosomes（2．5及5扯g）免疫小鼠后，接种L1210肿瘤细胞，有明显的抵抗肿瘤生长作用。结论：L1210分泌的exosomes在同基因小鼠模型中可产生较强的保护性免疫力，exosomes疫苗可作为白血病等恶性肿瘤免疫治疗的候选疫苗。

Objective：To investigate the anti-tumor immunity of the L1210 cell-secreted exosomes. Method：Exosomes were purified by ultrafiltration centrifugation and sucrose gradient uhracentrifugation. The expression of H-2D and ICAM-1 were investigated by solid phase immunoelectron microscopic method and the expression of HSPT0 was investigated by Western blot. DBA/2 mice were immunized with a given dose of exosomes （2.5 or 5 gg）. Tumor challenge experiments were performed by inoculation with L1210 leukemia cells. The exosomes elicited CTL-specific cytotoxicity was detected by MTT assay in vitro. Result： Transmission electron microscopy revealed that exosomes from L1210 cell culture supernatants were 50 to 100 nm membrane vesicles. The form was round or ellipse in shape. They were labeled by colloidal gold H-2D and ICAM-1. Immune electron microscopy showed the deposition of electron particles in the extra membrane of the vesicles. Western blot identified HSP70 in exosomes. Mice immunized by L1210 cell secreted exosomes could generate L1210 cell specific cytotoxic T lymphocytes （CTL）. Exosomes immunization partly inhibited the growth of implanted tumor in mice. There was only 15% and 40% tumorigenic rate in exosomes（5 μg） and （2.5 μg） immunized group respectively, while 100% in NS control group. Conclusion：Exosomes can be taken as a candidate of therapeutic vaccine for leukemia and other malignant tumors.