人外周血富集白细胞层来源的树突状细胞的培养与-第三军医大学学报.docVIP

人外周血富集白细胞层来源的树突状细胞的培养与鉴定【摘要】目的：探讨从人外周血富集白细胞层（白膜）分离、培养树突状细胞（Dendritic cell，DC）的方法。方法：用PBS液与白膜1：1稀释，采用Ficoll淋巴细胞分离液对稀释后的白膜进行淋巴细胞分离,2h后弃悬浮细胞,加入重组人粒细胞－巨噬细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素-4（rhIL-4）和DMEM完全培养液培养，体外培养第6天，加入肿瘤坏死因子（TNF-α）,促进其分化成熟。用流式细胞仪( flow cytometer, FCM)检测细胞表型. 在倒置显微镜下观察细胞形态。结果: 所获DC细胞表面CD83, CD86, CD1a, HLA-DR分子表达较高;倒置显微镜观察发现,细胞呈不规则形状,膜表面向外伸出许多树枝样细长突起. 结论: 我们建立了一种从白膜中分离、培养树突状细胞的方法,可从白膜中获得数量较多、纯度较高的DC。为DC瘤苗的来源开创了一条新途径，并为血液的合理利用找到了一个新方式。 【关键词】树突细胞；人外周血；富集白细胞层；细胞培养 【Abstract】 objective: To isolate and cultivate dendritic cell (DC) from human peripheral blood. Methods: The leukocyte-rich region separated from human peripheral blood was diluted 1:1 with phosphate buffered saline (PBS) and isolated mononuclear cells using Ficoll lymphocyte separating medium, the cells were incubated in DMEM supplemented with fetal calf for 2h, after which suspended cells were removed and fresh DMEM was added. Then recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human interleukin24 (rhIL24) were added into medium and the culture were incubated. Tumor necrosis factor-α(TNF-α) was added in 7days, Then the morphological features were observed by invert optical microscope.Finally, the phenotypes of cells were detected by flow cytometer and the morphs were by inverted microscope. Results: The levels of CD83, CD86, CD1a, and HLA-DR on cultured cells were higher than that of DCs. In addition, the cultured cells were irregular and slender synapses appeared on their surface. Conclusions: A method to isolate and cultivate DCs from human peripheral blood was successfully established, numerous and highly purified DCs were obtained in this way. This work contributes to a new way for exploration of DC-based tumor vaccine and reasonable utilization of blood. Key words: dendritic cell; peripheral blood; leukocyte-rich region, cell culture * 通信作者: 赵树铭，教授，主任医师，023shumingzhao@ 树突状细胞( Dendritic cell, DC) 由teinman 于1973年从小鼠淋巴结中分离成功，因其似树突状而得名[