IL-21单独或联合IL-15／IL-2对G-CSF动员人外周血单个核细胞体外增殖和抗肿瘤活性作用研究

本研究探讨IL-21单独或联合IL-15／IL-2对G—CSF动员的人外周血单个核细胞（G-PBMNC）体外增殖和抗瘤活性的影响，评价IL-21及相关细胞因子组合在肿瘤免疫治疗方面的可行性。应用IL-21单独或联合IL-15／IL-2体外培养G—PBMNC，用CCK-8法进行细胞增殖分析；以白血病细胞株K562为靶细胞。用CFSE／PI流式双染法检测其对肿瘤的杀伤作用；用流式细胞术分析免疫细胞表型。结果显示：培养72小时后单独IL-21因子组对靶细胞杀伤活性与IL-2结果相近；当效靶比为25：1时IL21＋IL15／IL21＋IL15＋IL2组与IL21＋IL2组相比杀伤能力有显著增强（P〈0．05）；效靶比50：1时，细胞因子联合应用组均显著高于细胞因子单独应用组（P〈0．05），以IL21＋IL15＋IL2诱导效果最佳。复苏冻存的G-PBMNC处理结果与新鲜的G—PBMNC结果基本-致。细胞因子组和对照组相比CD3、CD4和CD8抗原表达有所增加，以IL21＋IL15组的CD4、CD3^- 56^＋和CD3^＋56^＋抗原表达增加最为显著（P〈0．05）。结论：IL-21可增强G—PBMNC的体外杀伤活性，联合IL-15可更显著提高G—PBMNC的抗瘤活性。

Effects of IL-21 Alone or in Combination with IL-15/IL-2 on Proliferation and Anti-tumor Activity of G-CSF-Mobilized Peripheral Blood Mononuclear Cells In Vitro

This study was purposed to investigate the proliferation and antitumor activity of rhG-CSF-mobilized peripheral blood mononuclear cells （G-PBMNCs） activated by interleukin 21 （IL-21） alone or in combination with interleukin 15（ IL-15 ）/interleukin 2 （IL-2） and to evaluate the feasibility and value of tumor immunotherapy with cytokine combinations. G-PBMNCs were activated by IL-21 alone or in combination with IL-15/IL-2 in vitro, and the proliferation of the activated G-PBMNCs was analyzed by CCK-8 assay. The cytotoxicity of the activated G-PBMNC to the K562 cells was studied by the test principle which is based on target cell labeling with 5-（6）-carboxy-fluorescein succinimidyl ester （CFSE） and subsequent DNA-labeling with propidium iodide （PI） for identification of target cells with compromised cell membranes. The phenotypes of the activated G-PBMNCs were assayed by flow cytometry. The results showed that the cytotoxicity of IL-21 group had no difference from which of IL-2 group. When G-PBMNCs were exposed to the combinations of IL21 ＋ IL15/IL21 ＋ IL15 ＋IL2, the cytotoxicity was significantly enhanced at E： T ratio of 25： 1, as compared with combination of IL21 ＋ IL2 （p 〈 0.05 ）. The cytotoxicity of the cytokines combinations was significantly higher than that in cytokine used alone at E： T ratio of 50 ： 1 （p 〈 0. 05 ）. The cryopreservative and resuscitative G-PBMNCs showed the same result with the fresh G-PBMNCs in cytotoxicity test. The proportions of CD3^＋ and CD8^＋ T cells were increased when G-PBMNCs were incubated with cytokines for 72 hours. CD4, CD3^- 56 ^＋ and CD3 ^＋ 56 ^＋ counts were significantly elevated when G-PBMNCs were exposed to IL21 ＋ IL15 （p 〈 0.05 ）. It is concluded that IL-21 alone enhance the antitumor activity of G-PBMNCs, which further strengthens when IL-21 combinated with IL-15.