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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。
OBJECTIVE: To evaluate the effect of cytokine-induced killer (CIK) cells cultured with anti-CD3 monoclonal antibody I, L-2, IFN-γ and IL-1α in vitro. Methods: Peripheral blood mononuclear cells were collected from 8 patients with refractory lymphoma. Anti-human CD3 monoclonal antibody, human recombinant IL-1α and human recombinant IFN-γ were added on the first day of culture. Human recombinant IL- 2. Change the culture medium every 3 days and add recombinant human IL-2 and human recombinant IFN-γ to maintain the concentration. CIK cells were harvested from 12 to 14 days. Cell counts were performed on days 1, 4, 7, 10, and 13, and cellular immunophenotypes were measured before culture and on day 13 of culture. Results: After three days of co-stimulation with cytokines, colony formation occurred. After 13 days, the total number of CIK cells reached (7-18) × 109 (average 12.7 × 109), 44 ~ 140 times more than that before culturing The survival rates were over 90%. The mean values of immunophenotypic CD3 +, CD4 +, CD8 + and CD3 + CD56 + cells from pre-culture (50.9 ± 3.5)%, (29.9 ± 1.7)% and (41.3 ± 3.2) 1.6 ± 0.2% to (90.2 ± 1.6)%, (40.6 ± 5.5)% (, 52.8 ± 4.9)% and (33.1 ± 4.0)% on the 13th day of culture. CONCLUSION: IL-2 and IFN-γ levels are maintained during the culture after adding anti-human CD3 monoclonal antibody I, L-1α and IFN-γ on the day of culturing for 24 h and 24 h. CIK prepared by this four-factor culture system Cells in vitro amplification rate, tumor killing activity, and integrated peripheral blood easily available, easy to operate and self-source advantages, can be clinically applied.