树突状细胞体外培养体系的优化

Improvement of the culture system in vitro for dendritic cell

目的:探讨无血清专用培养基从人外周血单个核细胞(DC)分离培养树突状细胞的优化方法。方法:取正常成人新鲜血液,经密度梯度离心法,利用连续贴壁的方法获得单个核细胞,采用无血清培养基经人重组粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白介素-4(rhIL-4)和肿瘤坏死因子-α(TNF-α)诱导产生成熟DC,倒置显微镜观察树突状细胞结构,流式细胞仪检测DC表面标志物表达水平。结果:连续贴壁法无血清专用培养基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含10%胎牛血清RPMI培养基培养的DC。培养1周的DC高表达CD83、HLA-DR、CD80、CD86、CD83+HLA-DR+及CD80+CD86+。结论:采用无血清专用培养基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的DC。

Objective： To explore the fine method of isolated cultivation of dendritic cells （DC） from the peripheral blood. Methods- Utilizing successive adherence method to obtain the PBMCs, the fresh blood from normal adult was isolated by ficoll density gradient centrifugation. Adopting serum-free medium to induce the mature DC in vitro by recombinant human granulocyte-macrophage colony-stimulating factor （rhGM-CSF）, recombinant human interleukin-4 （rhIL-4） and tumor necrosis factor-α （FNF-α）. Then the morphological features were observed by invert optical microscope. Detecting the expression level of DC phenotypes by flow cytometry（FCM）. Observing the the morphological features of dendritic cells with invert optical microscope. Results： A large number of DCs with higher purity that were isolated from human peripheral blood in successive adherence method were obtained and compared with one time-adherence method. The DC cultured for one week highly expressed CD83,HLA-DR, CD80, CD86, CD83^＋HLA-DR^＋ and CD80^＋CD86^＋. Conclusion： Serum-free medium is adopted to get comparatively large number of DC with high purity in successive adherence method.