连续贴壁法分离培养人外周血来源树突状细胞及其超微结构的观察

Culturing dendritic cells from the peripheral blood with successive adherence method and observing the utralmicrostructure of cells

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.