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

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。

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

Background and purpose:Anthracycline can increase the immunogenicity of tumor cells.This study aimed to analyze the characteristics of mitoxantrone(MIT) treated B16F10-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 cells 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.