不同种培养基对体外诱导免疫细胞的生物活性及细胞毒作用

摘要

目的:探讨3种培养基对细胞因子诱导免疫细胞在增殖、免疫表型、杀伤、因子分泌等多方面生物学活性的影响.方法:正常人外周血单个核细胞分别用MD-CM-STM-N、AIM-V与RPMI 1640培养基经过CD3McAb、IL-2、IFN-γ诱导并培养.用台盼蓝活细胞计数计算细胞增殖情况,MTT法测定多种癌细胞杀伤,ELISA双抗夹心法测分泌IFN-γ的表达水平,流式细胞仪检测免疫表型.结果:与RPMI 1640组相比,MD-CM-STM-N和AIM-V组细胞快速增殖期延长至第13天.其中MD-CM-STM-N组最终扩增量最高,杀伤活性亦有明显提高(P＜0.05),CD8+、CD3+、CD56+的表达率也明显增加(P＜0.05)；细胞因子IFN-γ的分泌时间延长.结论:无血清培养基MD-CM-STM-N、AIM-V诱导免疫细胞的增殖能力、免疫表型表达率、细胞毒性、分泌细胞因子水平均高于RPMI 1640培养基,在增殖能力方面MD-CM-STM-N更具优势,为细胞因子诱导免疫细胞治疗提供了实验和理论依据.%Objective: To investigate the effects of three culture mediums on biological activity such as proliferation, phenotypes, killing effect and cytokine secretion. Methods: Peripheral blood mononuclear cells (PBMC) were cultured with C3McAb, IL-2 and IFN-γ in serum-free medium MD-CM-STM-N or AIMV or standard serum-containing RPMI 1640 medium separately. Proliferation, phenotypes and cytokine secretion of cells cultured in the three different medium were compared. The cellular proliferative capacity was evaluated by Trypan Blue living cell count, and so did efficiency in killing different tumor cells by MTT, the expression level of the secretory IFN-γ by ELISA, and phenotypes by flow cytometer (FCM). Results: Compared to cells cultured in standard serum-containing RPMI 1640 medium, the proliferation peaks of cells cultured in serum-free medium MD-CM-STM-N and AIMV were prolonged to the 13th day, and in the serum-free medium MD-CM-STM-N group ability of proliferation and the killing activity were significantly increased (P<0.05). More percentage of cells cultured in serum-free medium expressed CD8+, CD3+ and CD56+(P<0.05) , cells cultured in serum-free medium secreted more IFN- 7. Conclusion: The proliferative capacity, expression rate of phenotypes, cytotoxicity and cytokine secretion level of immune cells in the serum-free medium MD-CM-STM-N and AIMV are higher than those of them in standard serum-containing RPMI 1640 medium, and MD-CM-STM-N has the advantage of proliferative capacity which provides the experimental and theoretical basis for cytokine-induced immune cells therapy.