脐带血单个核细胞体外诱导成CIK细胞及其抗肿瘤效应的实验研究

Proliferation in vitro from the cord blood mononuclear cells and the anti-tumor effect of CIK cells

目的:探索建立CIK细胞的原代培养方 法并阐明其生物学特性,为该细胞在肿瘤生物过 继免疫治疗中的运用奠定基础。方法:通过加入 4种细胞因子(IFN γ、IL 2、IL 1及OKT3)将脐带 血单个核细胞(CBMNCs)诱导成CIK细胞;利用 流式细胞仪测定细胞表型、细胞增殖情况及细胞 周期的分布;利用改良的MTT法测定效应细胞 的体外细胞毒活性。结果:四种细胞因子的联合 运用,可诱导出大量的CIK细胞,其相对百分率 增长了163倍(从0.14%到22%),增殖高峰位 于第10～12天,针对K562、Hela和YTMLC肿瘤 细胞株,CIK细胞在效靶比为64∶1时的杀伤活 性分别为78.57±3.48、77.7±1.90和81.2± 1.51,在32∶1时分别为62.00±2.31、60.83± 2.80和64.07±2.87,在16∶1时分别为51.43± 2.51、52.87±2.91和54.10±3.11,均显著高于 LAK细胞及CBMNCs,P=0.001;而在效靶比为 8∶1(25.10±2.66、21.4±1.55和28.77±3.56) 及4∶1(17.10±2.10、11.93±1.86和9.49±2.92) 时与后两者差异无统计学意义,P=0.083。结 论:1)脐带血可作为诱导CIK细胞的重要来源 2)联合运用细胞因子能诱导出大量的CIK细胞 其增殖高峰位于培养的第12天,适宜在此期进 行临床运用;3)

OBJECTIVE:To investigate the protocol for the culture of CIK cells in vitro and so to build the experimental basement for their further clinical application. METHODS: CIK cells were generated by culture CBMNCs in the presence of four cytokines (IFN-γ,IL-2,IL-1 and OKT3). CIK cells were analyzed by FACS to confirm the phenotypes, proliferation and cellular cycles. The cytotoxicity in vitro was tested by the improved MTT assay. RESULTS:CIK cells were expanded significantly by co-culturing with the four cytokines. Their relative percentage was expanded 163 folds (from 0.14% to 22%). The proliferation peak appeared at day 10-12. At the ratio of 64∶1 between effective cells and target cells, CIK cells had the cytotoxicities of 78.57±3.48, 77.7±1.90 and 81.2±1.51 respectively against the tumor cell lines of K562, Hela and YTMLC, and at the ratio of 32∶1 the cytotoxicities were 62.00±2.31,60.83±2.80 and 64.07±2.87,at the ratio of 16∶1 they were 51.43±2.51 and 52.87±2.91 and 54.10±3.11, which were all significantly higher than LAK cells and CBMNCs,P=0.001. However at the ratios of 8∶1 (25.10±2.66,21.4±1.55,28.77±3.56)and 4∶1(17.10±2.10,11.93±1.86,9.49±2.92), there were no significant differences among them,P=0.083. CONCLUSIONS: 1) Cord blood can serve as an important materials for the generation of CIK cells; 2) CIK cells can be proliferated greatly after co-culturing with the four cytokines. It is a suitable time for clinical application at day 12,which is the proliferation peak;3) CIK cells possess higher cytotoxicity in vitro than LAK cells, and so using them reasonably will provide a new hope for tumor’s adoptive cellular immunotherapy.