米托蒽醌处理B16F10-ESAT-6-gpi/IL-21瘤苗特征及其诱导的抗肿瘤免疫反应初探

The characteristics of the B16F10-ESAT-6-gpi/IL-21 tumor vaccine inactivated by mitoxantrone and the preliminary study of its anti-tumor immune responses

背景与目的:蒽环类药物处理可使肿瘤细胞免疫原性增加。本文旨在分析米托蒽醌(mitoxantrone,MIT)处理的B16F10-ESAT-6-gpi/IL-21瘤苗特征,初步探讨该瘤苗诱导的抗肿瘤免疫反应。方法:MIT处理B16F10-ESAT-6-gpi/IL-21瘤苗后,用吖啶橙/嗅化乙啶(AO/EB)染色法观察瘤苗细胞形态,流式细胞仪(FCM)检测其粒度及凋亡比例,荧光显微镜观察瘤苗凋亡后细胞膜表面结核杆菌早期分泌靶抗原6KD(ESAT-6)的表达情况,蛋白质印迹法(Western blot)检测瘤苗经MIT处理后IL-21的表达。瘤苗免疫小鼠后,FCM检测了补体依赖的细胞毒性(complement dependent cytotoxicity,CDC)及细胞毒T细胞(cytotoxicT lymphocyte,CTL)活性。结果:经MIT处理后,瘤苗停止分裂,细胞逐渐增大,数日内可保持生物活力,并表达IL-21。瘤苗细胞凋亡后,ESAT-6成点簇状分布于胞膜表面。MIT处理的瘤苗能诱导小鼠产生抗肿瘤免疫应答,免疫鼠血清和CD8+T细胞可分别通过CDC和CTL杀伤野生型B16F10细胞。结论:MIT处理的B16F10-ESAT-6-gpi/IL-21瘤苗失去增殖能力,但仍能表达IL-21且具有免疫原性,能诱导小鼠产生抗肿瘤免疫反应。

Background and purpose： Anthracycline can increase the immunogenicity of tumor cells. This study aimed to analyze the characteristics of mitoxantrone （MIT） treated B 16F 10-ESAT-6-gpi/IL-21 tumor vaccine and primarily investigate its anti-tumor immune effect induced in mice. Methods： After treating tumor vaccine with MIT, the cell morphology was observed by AO/EB staining, and the particle size and apoptosis ratio were detected by flow cytometry （FCM）. The expression of ESAT-6 in the apoptotic tumor vaccine was observed through immunofluorescence microscope, and the expression of IL-21 in MIT treated tumor vaccine was detected by Western blot. The immune effect of tumor vaccine was evaluated by animal experiment, and the complement dependent cytotoxicity （CDC） in vitro was detected through FCM. The cytotoxic T lymphocyte （CTL） activity was analyzed by CFSE/7-AAD cytotoxic experiment. Results： The tumor vaccine treated with MIT ceased cell division, the cells gradually enlarged, maintained biological activity and expressed IL-21 in several days. After cell apoptosis, ESAT-6 was expressed on the surface of cell membrane in clusters. Murine anti-tumor immunity could be induced by MIT treated tumor vaccine. The immune serum and CD8＋T cells in mice could kill wild type B16F10 ceils by CDC and CTL cytotoxicity, respectively.Conclusion： MIT treated B16F10-ESAT-6-gpi/IL-21 tumor vaccine could maintain immunogenicity and induce anti- tumor immune responses though the vaccine loses the proliferative capacity.