胃癌患者外周血树突状细胞的体外扩增、鉴定及内吞能力测定

Proliferation,identification,and endocytosis ability of dendritic cells from peripheral blood of gastric cancer patients

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 study the proliferation, identification and endocytosis ability of dendritic cells from the peripheral blood of gastric cancer patients. Methods The mononuclear cells isolated from peripheral blood in the patients with gastric cancer were cultured in RPMI medium 1640 containing reconstructive human granulocyte macrophage colony-stimulating factor（rhGM-CSF）, IL- 4, and TNF-α to generate dendritic cells. The morphologic characteristics of these cells were observed with light microscope and the phenotypic figures were analysed with FACS. The function of the cells was reflected in mixed lymphocyte reaction assay （MLR） and endocytosis ability was detected by the horseradish peroxidase （HRP） uptake experiment. Results Peripheral blood mononuclear cells （PBMNCs） were cultured in present of rhGM-CSF, IL-4, and TNF-α for 9 days, these cells bear typical morphologic and phenotypic characteristics of dendritic cells, expressed MHC Ⅱ （HLA-DR） （69.70±6.12）%, CD80（65.32±5.19）%, CD86（71.65±7.61）%, CD1a（58.65±3.28）% and CD14（16.82±1.25） %, gained the powerful capacity of stimulating allogeneic T cells. It showed the characteristics of mattire DCs. In addition, these cells had active endocytosis ability, which peaked on the sixth day in culture but deceased afterwards. Conclusion This is a reliable method to induce and proliferate DCs, which may be a promising candidate in new biotherapy strategies against gastric cancer.