白细胞介素12锚定修饰肿瘤源性外核体肾癌疫苗的制备及其体外抗肿瘤效应

Preparation of renalcancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro

目的 制备白细胞介素12（IL-12）锚定修饰肿瘤源性外核体（EXOs）的肾癌疫苗,探讨其体外对肾癌细胞特异性杀伤效应.方法 将精基化磷脂酰肌醇（GPI）信号肽序列与IL-12基因融合,构建真核双表达质粒pBIG,应用激光共聚焦显微镜和流式细胞仪检测融合蛋白（GPI-IL-12）在肾癌细胞中的表达.用超滤和蔗糖/重水密度梯度离心法分离EXOs,透射电镜鉴定其形态.采用Western blot检测其标志性分子热休克蛋白70（HSP70）、细胞间黏附分子（ICAM-1）、肾癌特异性抗原G250和GPI-IL-12的表达.应用酶联免疫吸附试验（ELISA）测定EXOs负载IL-12的量,干扰素γ（IFN-γ）释放试验检测IL-12修饰EXOs（EXO-IL-12）的功能,羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）和碘化丙啶（PI）双标的流式细胞仪分析其对肾癌细胞的诱导杀伤效应.结果 EXOs为类圆碟形、双层膜结构,直径30～80nm,表达HSP70、ICAM-1、G250和GPI-IL-12.ELISA测定结果显示,10μg/ml EXOs含约（80.0±9.6）pg/ml的IL-12.EXO-GPI-IL-12疫苗能显著地促进T淋巴细胞分泌IFN-γ,在体外对肾癌细胞的特异性杀伤率为53.7%,明显高于对膀胱癌细胞（9.8%）和结肠癌细胞的杀伤率（10.8%）,差异有统计学意义（P〈0.01）.结论 EXO-GPI-IL-12疫苗能够在体外诱导T淋巴细胞产生较强的抗原特异性细胞毒作用,可望成为治疗肾癌的新疫苗.

Objective To prepare a vaccine of IL-12-anchored exosomes derived from renalcancer cells and to evaluate its antitumor effect in vitro. Methods A mammalian co-expression plasmid of glycolipid-anchor-IL-12 （GPI-IL-12） was constructed by subcloning IL-12A chain gene （P35 subunit） and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene （P40 subunit） in pBudCE4. 1.Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-γ release assay. Results Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. （80.0±9.6）pg/ml of IL-12 was detected in 10μg/ml exosomes and it significantly induced the release of IFN-γ. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes （CTL）,resulting in more significant cytotoxic effects in vitro. Conclusion A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.