趋化因子巨噬细胞炎症蛋白-1α动员的树突状细胞经基因修饰后抗胃癌效应

Anti-gastric carcinoma efficacy of gene modified dendritic cell vaccine recruited by macrophage inflammation protein-1

目的观察趋化因子巨噬细胞炎症蛋白-1α(MIP-1α)体外动员的树突状细胞(DC)经基因修饰后在荷瘤小鼠体内的抗胃癌效应。方法615小鼠通过尾静脉注射MIP-1α,流式细胞仪分选出B220^- CD11c^+细胞,加入鼠粒-巨噬细胞集落刺激因子(mGM-CSF)、白细胞介素- 4(IL-4)和鼠肿瘤坏死因子-α(mTNF-α)贯续培养进行诱导分化。通过细胞表型和混合淋巴细胞反应,检测细胞因子培养前后B220^- CD11c^+细胞的异同。收集培养后的B220^- CD11c^+细胞,加入编码黑色素瘤抗原基因-3(MAGE-3)的重组腺病毒进行转染,制备表达肿瘤抗原的DC疫苗。制备小鼠前胃癌(MFC)实体瘤模型,DC疫苗于MFC细胞接种后经皮下注射,观察小鼠瘤体生长和存活情况,研究DC疫苗的免疫治疗作用。结果MIP-1α注射8h后外周血中B220^- CD11c^+细胞数量即升高,48h达到高峰,占外周血单个核细胞(MNCs)(13.68±0.95)%。新鲜分离的B220^- CD11c^+细胞不具有成熟DC的特征,而经体外细胞因子诱导分化后则具有典型的DC表型,并具有极强的刺激T细胞增殖的能力。小鼠皮下接种MFC细胞后注射基因修饰的DC疫苗,小鼠瘤体生长缓慢,存活时间明显延长,与对照组之间差异有统计学意义(P<0.01)。结论注射趋化因子MIP-1α可快速动员B220^- CD11c^+细胞进入小鼠外周血,并经细胞因子可诱导分化为成熟DC。MIP-1α动员的DC经基因转染制备的DC疫苗,在体内对荷瘤小鼠有明显的免疫治疗作用,且较荷载全肿瘤细胞抗原的DC疫苗作用明显增强。

Objective To study dendritic cells （DC） precursors that were mobilized into the peripheral blood by injection of macrophage inflammation protein-1α （ MIP-1α ） and the anti-tumor effects induced by MIP-1α mobilized DC vaccine expressing tumor antigen in vivo. Methods 615 mice were injected with MIP-1α via the tail vein. B220^- CD11c^＋ cells were sorted from the peripheral blood mononuclear cells （MNCs） by FACS. The quantitive change of B220^- CD11c^＋ cells were compared among MNCs. Freshly isolated B220^- CD11c^＋ cells and B220^- CD11c^＋ cells cultured with mouse granulocytemacrophage colony-stimulating factor （ mGM-CSF）, interleukin 4 （ IL-4）, and mouse tumor necrosis factor-1α （mTNF-1α） were analyzed by phenotype analysis,and mixed lymphocyte reaction （MLR）. For adenoviral （Ad）-mediated gene transduction, cultured B220^- CD11c^＋ cells were incubated with Ad-melanoma antigen gene-3. B220^- CD11c^＋ cells pulsed mouse forestomach carcinoma cells （MFC） tumor lysate was used as control. To establish the solid tumor model,groups of 615 mice were injected with MFC cells subcutaneously in the abodminal wall. DC vaccines were immunized after the challenge of MFC cells, then the tumor size and the survival of mice which could detect the anti-tumor effects of DC vaccines were observed. Results B220^- CD11c^＋ cells were increased in the circulation 8 h after MIP-1α injection, then gradually reached a peak level 48 h after injection,accounting for （ 13.68 ±0.95） % among MNCs. Freshly isolated B220^- CD11c^＋ cells did not show the characters of mature DC, while cultured B220^- CD11c^＋ cells were phenotyicaUy identical to typical DC and gained the capacity to stimulate aUogeneic T cells. These MIP-1α mobilized DCs were transduced with Ad-MAGE-3, which were prepared for DC vaccines expressing tumor antigen. Five days after the challenge of MFC cells ,mice were subsequently injected with DC vaccines. The tumor size in the experimental group was much smaller than that in the control groups 27 days after the challenge. Kaplan-Meier survival curves revealed that the survival of the mice in the experimental group was much longer than that in the control groups （P 〈0.01 ）. Conclusion B220^- CD11c^＋ DC precursors were rapidly accumulated in the peripheral blood after injection of MIP-1α in mice, which could further differentiate into mature DCs. These MIP-1α mobilized DCs, when transduced with MAGE-3 gene,could generate therapeutic anti-tumor effects on MFC cells loading mice in vivo. The efficiency of anti-tumor immunity induced by MIP-1α mobilized DCs expressing tumor antigen were much more potent than MIP-1α mobilized DCs pulsed MFC cells tumor lysate.