摘要目的 探讨IL-21联合IL-6对脐血来源的CIK细胞杀伤K562及HL-60白血病细胞系效应的影响.方法 Ficoll法分离脐血单个核细胞,加入细胞因子诱导培养CIK细胞并分为4组:对照组,IL-21组,IL-6组,IL-21 +IL-6联合组.流式细胞术检测CIK细胞免疫表型及Treg细胞的表达;CCK-8法检测细胞的增殖活性;LDH释放法检测CIK细胞对K562及HL-60细胞的杀伤作用.结果 在细胞培养的第7天,与对照组相比,IL-21组CIK细胞的产生由(7.30±1.20)％上升至(12.52±1.45)％,IL-6组升至(11.01±1.04)％,IL-21 +IL-6组升至(20.80±2.33)％,而IL-21组Treg细胞表达由(26.18±1.03)％降至(10.95±1.49)％,IL-6组降至(17.91±1.95)％,IL-21 +IL-6组降至(5.25±0.54)％;在第14天,IL-21与IL-6组的CIK细胞比例为对照组的2倍,IL-21 +IL-6组为对照组的2.5倍,而Treg细胞比例在IL-21组由(9.24±0.42)％降至(1.48±0.06)％,IL-6组降至(3.96±0.11)％;IL-21 +IL-6组Treg细胞降至(0.83±0.08)％.在效靶比为20∶1时,对照组细胞对K562及HL-60细胞的杀伤率[(29.31±0.58)％、(20.14±1.27)％]均明显低于各实验组(P＜0.01),而IL-21 +IL-6联合组对两种细胞的杀伤率[(63.79±0.91)％、(41.53士1.43)％]均高于IL-21组[(52.99±1.26)％、(33.04±1.68)％]和IL-6组[(53.05±1.52)％、(32.18±3.83)％](P＜0.01),IL-21与IL-6组间差异无统计学意义.结论 IL-21和IL-6可增强脐血来源的CIK细胞增殖与杀伤活性,同时可显著降低Treg细胞的表达,从而间接增强CIK细胞的功能;而且,二者协同使用效果更强,使CIK细胞对K562及HL-60白血病细胞系发挥更强大的杀伤作用,具有潜在的临床应用价值.

abstractsObjective To explore the effects of interleukin 21 (IL-21) incombinetion with interleukin 6(IL-6) on cytotoxic activity of cytokine induced killer(CIK) cells derived from umbilical cord blood on K562 and HL-60 cells.Methods Mononuclear cells were separated from fresh umbilical cord blood by ficoll-hypaque density centrifugation and cultured with cytokines to induce CIK cells.Cells were then divided into four groups including IL-21 treated group,IL-6 treated group,IL-21 +IL-6 treated group and untreated control group.Immunophenotypes of CIK and Treg cells weredetected by flow cytometry.The proliferation of CIK cells in each group was measured by CCK-8 assay.Cytotoxic activities of CIK cells from each treatment group on K562 and HL-60 cells were detected by LDH release assay.Results As compared with control group,on day 7 after cytokine treatment,the proportion of CD3+CD56+CIK cells in IL-21 treated group was increased from (7.30±1.20)％ to (12.52±1.45)％,and up to (11.01±1.04)％ in IL-6 group,(20.80±2.33) ％ in IL-21 +IL-6 group respectively.However,Treg cells in IL-21 treated group was decreased from (26.18±1.03)％ to (10.95±1.49)％,and down to (17.91±1.95)％ in IL-6 treated group,(5.25± 0.54) ％ in IL-21+IL-6 treated group,respectively.On day 17,the proportion of CIK cells in IL-21 and in IL-6 treated group was 2 times higher than that in control group.Furthermore,the proportion of CIKs was elevated up to 2.5 times in IL-21 +IL-6 treated group as compared with control group.While the percentage of Treg cells in IL-21 treated group was decreased from (9.24±0.42)％ to (1.48±0.06)％,and (3.96± 0.11)％ in IL-6 treated group,Treg cells was decreased to (0.83±0.08)％ in IL-21 +IL-6 treated group.The cytotoxic activity of CIK cells from control group on K562 and HL-60 cells at the ratio of 20 ∶ 1 were (29.31±0.58)％ and (20.14±1.27)％ respectively,which was significantly lower than that in cytokine treated groups (P＜0.01).While cytotoxic activities of CIKs from IL-21 +IL-6 treated group [(63.79± 0.91) ％,(41.53± 1.43) ％] were higher than that in IL-21 treated group [(52.99± 1.26) ％,(33.04± 1.68)％] and IL-6 treated group [(53.05±1.52)％,(32.18±3.83)％] (P＜0.01),there were no difference between IL-21 treated group and IL-6 treated group.Conclusion IL-21 and IL-6 could enhance the proliferative and cytotoxic activity of CIK cells.The significant reduction in proportion of Treg cells in response to cytokine treatment might contribute to an increased cytotoxic activity of CIK cells.Combined use of IL-21 and IL-6 stimulated strongest cytotoxic activity of CIKs on K562 and HL-60 cells,suggesting a possibility of clinic application.