重组人粒细胞集落刺激因子动员后人外周血单核细胞来源树突状细胞肿瘤疫苗治疗研究

Recombinant human granulocyte colony-stimulating factor mobilized human peripheral blood monocytes derived dendritic cell vaccine as a tumor immunotherapy

目的树突状细胞(dendritic cell,DC)疫苗是肿瘤免疫治疗的一个重要手段,在细胞技术层面对其优化是当前研究的热点。文中探讨了重组人粒细胞集落刺激因子(recombinant human granulocyte colony-stimulating factor,rhG-CSF)动员后外周血单核细胞(monocyte,Mo)来源DC疫苗临床应用可行性。方法采取回顾性的方法分析2011年8月至2011年12月行Mo-DC疫苗治疗的59例肿瘤患者,其中32例进行了采集前的rhG-CSF动员,同期27例未动员患者作为对照。动员组在血细胞分离机采集前行rhG-CSF皮下注射(1.25μg/kg×1d或×2d或×3d),动员前后均检测血常规;分析rhG-CSF动员组和对照组采集效率、采集终产品中单个核细胞(mononuclear cells,MNC)数量及活力变化;比较相同采集循环量下2组Mo-DC细胞计数、得率、纯度及存活率。流式细胞术(FCM)检测动员组和对照组Mo-DC细胞表型,酶联免疫吸附法(ELISA)检测细胞上清中细胞因子白介素12(interleukin-12,IL-12)、干扰素γ(interferon-γ,IFN-γ)和IL-10浓度。结果 rhG-CSF动员3 d后外周血Mo计数与动员前相比上升明显,差异有统计学意义(P<0.05);rhG-CSF动员组和对照组采集效率、血小板丢失率和采集终产品中MNC活力差异无统计意义,但相同采集循环量时rhG-CSF动员3 d后采集终产品中MNC数量与对照组差异有统计学意义(P<0.05)。相同采集循环量时rhG-CSF动员组DC产量明显高于对照组(P<0.05),但DC细胞得率、纯度和存活率差异无统计意义。rhG-CSF动员组和对照组DC细胞HLA-DR、CD11c、CD80、CD86、CD83及CD54表达差异无统计意义,DC细胞培养上清中IFN-γ、IL-12和IL-10表达差异亦无统计意义。结论 Mo-DC疫苗治疗的肿瘤患者采集前进行至少3 d的rhG-CSF动员可以有效提升DC疫苗产量,有助于提高疫苗治疗疗效。

Objective The purpose of this study was to investigate the feasibility of clinical application of recombinant human granulocyte colony-stimulating factor （rhG-CSF）-mobilized monocytes （Mo） derived dendritic cell （DC） vaccine. Methods We retrospectively nanalyzed 59 cases of tumor treated by Mo-DC vaccine therapy, of whom 32 were mobilized with rhG-CSF, and the other 27 unmobilized as controls. For the mobilized group, rhG-CSF was injected subcutaneously （ 1.25 μg/kg/24 h × 1 d or × 2 d or × 3 d） before apheresis using a blood cell separator, and blood routine examinations were performed before and after mobilization. Comparisons were made between the mobilized and control groups in collection efficiency and the number and viability of mononuclear cells in the final apheresis products, as well as in the count, yield rate, purity and viability of Mo-DCs under the same collection circu- lating volume. The phenotypes of the Mo-DCs were determined by FCM and concentrations of cytokines IL-12, IFN-γand IL-10 in the cell culture supematants were measured by ELISA. Results After 3 days of rhG-CSF mobilization, the Mo count in the peripheral blood was significantly increased as compared with the baseline （P 〈 0. 05）. There were significant differences between the mobilized and control groups in the number of mononuclear cells in the final apheresis products （P 〈 0. 05）, but not in the collection efficiency, platelet loss viability of mononuclear cells. The Mo-DC yield was significantly higher in the mobilized group than in the control （ P 〈 0. 05）, but no significant differences were found in the Mo-DC yield rate, purity and viability between the two groups, nor in the expressions of HLA-DR, CDllc, CD80, CD86, CD83 and CD54, and in the concentrations of cytokines IL-12, IFN-γ/and IL-10 in the cell culture supernatants. Conclusion rhG-CSF mobilization for tumor patients receiving Mo-DC vaccine therapy for at least 3 consecutive days before apheresis could efficiently improve the Mo-DC vaccine yield and hence the outcome of vaccine therapy.