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目的探讨胃癌患者外周血诱导扩增树突状细胞(DCs)的方法,观察其形态、表型、内吞及递呈抗原能力。方法从胃癌患者外周血中分离单个核细胞(PBMNCs),在重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的诱导下培养扩增树突状细胞。光学显微镜观察其体外培养过程中的形态特征,流式细胞仪(FACS)分析其表型特征,混合淋巴细胞反应(MLR)检测其功能,辣根过氧化酶(HRP)内吞实验检测其群体内吞能力。结果PBMNCs经过rhGM-CSF、IL-4和TNF-α诱导培养9d后,具典型的DCs形态,细胞表型为主要组织相容性复合体-2(MHCⅡ)类分子(HLA-DR)(69.70±6.12)%、CD80(65.32±5.19)%、CD86(71.65±7.61)%、CD1a(58.65±3.28)%和CD14(16.82±1.25)%,具有极强的激发MLR的能力,显示成熟DCs的特征。DCs群体的内吞能力约在培养第6天达高峰,之后有明显下降。结论该方法是一种可靠的诱导、扩增DCs的方法,在其具有活跃的内吞能力时,肿瘤抗原致敏可作为胃癌生物治疗的手段之一。
Objective To investigate the method of inducing dendritic cells (DCs) in peripheral blood of patients with gastric cancer and to observe their morphology, phenotype, endocytosis and antigen presenting ability. Methods Mononuclear cells (PBMNCs) were isolated from the peripheral blood of patients with gastric cancer, and the effects of rhGM-CSF, IL-4 and TNF- α) to induce dendritic cells. Morphological characteristics during in vitro culture were observed by optical microscopy. The phenotypic characteristics were analyzed by flow cytometry (FACS), the function of mixed lymphocyte reaction (MLR) and the endocytosis of horseradish peroxidase (HRP) Endocytic capacity. Results After treated with rhGM-CSF, IL-4 and TNF-α for 9 days, PBMNCs exhibited typical morphology of DCs. The phenotype of PBMNCs was MHC class II (HLA-DR) (69.70 (6.12 ± 5.19)%, CD86 (71.65 ± 7.61)%, CD1a (58.65 ± 3.28)% and CD14 (16.82 ± 1.25)%, respectively, with extremely strong ability to stimulate MLR feature. The endocytosis ability of DCs reached a peak on the 6th day of culture, and then decreased significantly. Conclusion This method is a reliable method for inducing and amplifying DCs. When it has active endocytosis, tumor antigen sensitization can be used as one of the means of biological treatment of gastric cancer.