目的:改进人外周血单个核细胞分离培养树突状细胞的方法,电镜观察树突状细胞超微结构。方法:利用连续贴壁法分离获得CD14^+前体细胞,经人重组粒细胞/巨噬细胞集落刺激因子(rhGM—CSF)、白介素-4(rhIL-4)和肿瘤坏死因子α(TNF—α)诱导产生成熟DC,流式细胞仪检测DC表面标志物表达水平,透射电镜(TEM)观察树突状细胞超微结构。结果:连续贴壁法体外分离培养人外周血DC,所获得DC数量和纯度均都多于以往一次贴壁分离培养的DC。培养1周的DC高表达CD1a、CD40、HLA—DR、CD80、CD86。透射电镜观察到不同状态树突状细胞的超微结构。结论:连续贴壁法可分离培养数量较大、纯度较高的人外周血来源DC。
Objective: To improve the method of isolated cultivation of dendritic cells (DC) from the peripheral blood and observe the utralmicrostructure of dendritic cells with electron microscope, Methods: Utilizing successive adherence method to obtain CD14^+ precursor cells, The mature DC was induced in vitro by granuloeyte - macrophage colony - stimulating factor ( GM - CSF), interleukin - 4 ( IL - 4 ) and tumor necrosis factor - α ( TNF - α ). Detecting the expression level of DC surface markers by flow cytometry. Observing the utralmicro structure of dendritic cells with transmission electron microscope (TEM). Results: A large number of of DC with higher purity that were isolated from human peripheral blood in successive adherence method were obtained compare with one time - adhenrence method. The DC cultured one week highly expressed CD1 a, CD40, HLA - DR, CD83 and CD86. We observed the utralmicrostructure of dendritic cells in different state with transmission' electron microscope ( TEM ). Conclusion: We can get comparatively large number of DC with high purity in successive adherence method.