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目的研究膀胱癌患者外周血树突状细胞(DC)的体外培养扩增和鉴定。方法应用Ficoll-Hypaque离心获得界面细胞,贴壁培养2h,获得单个核细胞,体外以重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF,50ng/ml)+重组人白细胞介素4(rhlL-4,10ng/ml)+人肿瘤坏死因子-α(hTNF-α,50ng/ml)诱导培养。倒置相差显微镜及扫描电镜下观察DC生长情况；流式细胞仪检测DC表型；MTT法检测DC活化的T细胞对肿瘤细胞BIU87的杀伤率。结果第6天体外培养的DC由贴壁状态变为悬浮毛刺状细胞,第8天为形态不规则的毛刺状,为典型DC形态。流式细胞仪检测表明,成熟DC高水平表达CD_(1a)、CD_(83)、CD_(86)及HLA-DR等。被DC激活的T细胞对膀胱癌细胞株BIU87的杀伤率为(48.8±3.7)%,未经DC激活的T细胞的杀伤率为(25.7±1.5)%,2组比较差异有统计学意义(P＜0.01)。结论膀胱癌患者外周血经rhGM-CSF+rhIL-4+hTNF-α体外诱导培养能诱导出DC。
Objective To study the expansion and identification of peripheral blood dendritic cells (DCs) in bladder cancer patients in vitro. Methods Interfacial cells were isolated by Ficoll-Hypaque centrifugation and cultured for 2 hours. The mononuclear cells were obtained and cultured in vitro. Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF, 50 ng / ml) and recombinant human interleukin- rhlL-4,10ng / ml) + human tumor necrosis factor-α (hTNF-α, 50ng / ml). The growth of DC was observed under inverted phase contrast microscope and scanning electron microscope. The DC phenotype was detected by flow cytometry. The killing rate of DC-activated T cells to BIU87 was detected by MTT assay. Results On the 6th day, DC cultured in vitro changed from adherent state to floating hairy cells. On the 8th day, the shape of irregular DC was burr, which was the typical form of DC. Flow cytometry showed that CD_ (1a), CD_ (83), CD_ (86) and HLA-DR were highly expressed in mature DCs. The killing rate of DCs activated by DC was (48.8 ± 3.7)% in bladder cancer cell line BIU87 and (25.7 ± 1.5)% in non-DC activated T cells. The differences between the two groups were statistically significant ( P <0.01). Conclusion The DCs can be induced in vitro by rhGM-CSF + rhIL-4 + hTNF-α in bladder cancer patients.