细胞因子诱导的杀伤细胞体外培养技术的评价

目的:评价采用抗CD3单抗I、L-2、IFN-γ和IL-1α四因子培养体系体外培养细胞因子诱导的杀伤细胞(CIK细胞)的效果。方法:采集8例难治性淋巴瘤患者外周血单个核细胞培养,于培养第1天加入抗人CD3单抗、人重组IL-1α、人重组IFN-γ,第2天加入人重组IL-2,每3天换培养液并添加人重组IL-2和人重组IFN-γ以维持其浓度,培养12~14 d收获CIK细胞。于培养第1、4、7、10、13天进行细胞计数,于培养前及培养第13天测定细胞免疫表型。结果:细胞因子共刺激3 d即出现细胞集落,13 d后CIK细胞总数达(7~18)×109(平均12.7×109),较培养前扩增44~140倍(平均98倍),细胞存活率超过90%;具有免疫表型CD3+、CD4+、CD8+和CD3+CD56+细胞的平均值分别从培养前的(50.9±3.5)%、(29.9±1.7)%、(41.3±3.2)%(、1.6±0.2)%增加到培养第13天的(90.2±1.6)%、(40.6±5.5)%(、52.8±4.9)%、(33.1±4.0)%。结论:培养当日加抗人CD3单抗I、L-1α、IFN-γ,24 h后加IL-2,培养过程中维持IL-2和IFN-γ浓度,这种四因子培养体系制备的CIK细胞的体外扩增率高、杀瘤活性强,并综合了外周血易获得、操作简便和自体来源等优点,可以临床推广应用。

Preliminary Study on the Culture Method of Killer Cells Induced by Cytokines

objective:To explore the method to culture killer cells induced by cytokines.Methods:Peripheral blood mononuclear cells from 8 patients with refractory lymphoma were collected.The CIK cells were cultured with various cytokines(IFN-γ,IL-1α,IL-2 and CD3 mAb) at different time points during the culture process.The immune phenotypes of the heterogeneous cells were determined.Results:CIK cells stimulated by cytokines(IFN-γ,IL-1α,IL-2 and CD3 mAb) have proliferated rapidly,and increased about 44～140 times at day 13.The average percentage of cells harboring surface marker of CD3+、CD4+、CD8+ and CD3+CD56+ were also increased to(90.2±1.6)%、(40.6±5.5)%、(52.8±4.9)% and(33.1±4.0)% 2～3 weeks after cytokines stimulation.Conclusion:Exogenous cytokines contribute to proliferation of CIK cells in vitro.This method could be applied clinically because of the simplicity of the operation and autogenic source of the cells.