脐血树突状细胞与细胞因子诱导的杀伤细胞协同抗急性白血病细胞效应及生物学活性研究

Synergic effects of cord blood DCs and homologous CIK cells on acute leukemia cells and their biological activities

目的研究脐血树突状细胞-细胞因子诱导的杀伤(DC-CIK)细胞的体外增殖、免疫表型、分泌细胞因子水平及其对急性白血病细胞细胞毒作用的影响。方法采集脐血单个核细胞诱导DC和CIK细胞。将DC和CIK细胞按1∶5的比例混合培养,以脐血CIK细胞或外周血DC-CIK细胞为对照。用流式细胞术分析细胞表型,台盼蓝活细胞计数计算细胞扩增倍数,MTT法检测效应细胞杀伤白血病细胞的活性,ELISA法测定分泌干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白细胞介素-12(IL-12)的水平。结果脐血DC-CIK细胞的增殖能力显著高于脐血CIK细胞和外周血DC-CIK细胞(P均<0.05);脐血DC、CIK细胞共培养后,CD3+CD8+、CD3+CD56+细胞比例较同条件下CIK细胞明显增多(P<0.05);混合培养3 d,脐血DC-CIK细胞上清液中IL-12、IFN-γ、TNF-α含量均比CIK细胞单纯培养的分泌量高(P<0.01或P<0.05);在2.5∶1～20∶1的效靶范围内,脐血DC-CIK细胞对各亚型急性白血病细胞的杀伤率明显高于CIK细胞(P<0.05),且对各亚型白血病细胞杀伤活性无统计学意义,与外周血DC-CIK细胞对白血病杀伤效应相类同。结论脐血DC可增强同源CIK细胞的增殖活性和抗白血病效应。脐血DC-CIK细胞增殖能力比外周血DC-CIK细胞强,但两者在细胞毒方面无显著性差异。脐血来源丰富,且输注不易引起严重的移植物排斥反应,其DC-CIK细胞在免疫治疗方面应有更广泛的临床应用前景。

Objective To study the proliferation capability in vitro,immunophenotype changes and secreted cytokines level of cord blood DC-CIK cells and their effect on cytotoxicity of acute leukemia cells.Methods DCs and CIK cells were induced from cord blood mononuclear cells.They were co-cultured according to the ratio of 1∶5,and CIK cells from cord blood or DC-CIK cells from peripheral blood were cultured as controls.Immunophenotype changes were analyzed by flow cytometry,the increased number of cells was counted by trypan-blue staining,and the killing effect on leukemia cells was detected by MTT assay.The interferon-γ（IFN-γ）,tumor necrosis factor-α（TNF-α） and interleukin-12（IL-12） levels of the cultured supernatants were detected by ELISA kits.Results The proliferation capability of cord blood DC-CIK cells was significantly higher than that of cord blood CIK cells and peripheral blood DC-CIK cells（both P0.05）.Under the same condition,the ratio of double positive cells CD3＋CD8＋ and CD3＋CD56＋ in CIK cells was significantly enhanced when co-cultured with cord blood DC cells（P0.05）.The levels of IL-12,IFN-γ and TNF-α in cultured supernatants of cord blood DC-CIK cells increased noticeably on day 3 as compared with those of CIK cells cultured alone（P0.01,P0.05,P0.05）.Within the effector-target ratio range from 2.5∶1 to 20∶1,the killing capacity of cord blood DC-CIK cells against all subtypes of acute leukemia cells was much higher than that of CIK cells（P0.05）,but there was no significant difference among all subtypes of acute leukemia cells.It was the same as the killing effect of peripheral blood DC-CIKcells on leukemia cells.Conclusion The proliferation capability and anti-leukemia effect of the homologous CIK cells can be enhanced by cord blood DCs.The proliferation capability of cord blood DC-CIK cells is stronger than that of peripheral blood DC-CIK cells,but there is no significant difference in cytotoxicity between them.Because cord blood can be easily obtained and is not likely to cause serious graft rejection,DC-CIK cells have more extensive clinical application in immune therapy.