中文摘要：

目的:建立小鼠骨髓源性树突状细胞(DCs)体外培养和扩增方法,为以DCs为靶点的免疫治疗研究提供基础。方法:从C57BL/6小鼠股骨和胫骨中提取骨髓细胞,以含重组小鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF,400U/ml)和IL-4(200U/ml)的RPMI 1640(含10%FBS)进行诱导培养,于培养第4天加入脂多糖(LPS)继续培养2d。利用倒置相差显微镜和透射电子显微镜观察细胞生长状态和超微结构,流式细胞术鉴定培养第6天时DCs纯度及免疫表型,流式细胞微球捕获芯片技术检测第2、4、6天培养上清中细胞因子IL-6、IL-10、巨噬细胞趋化因子-1(MCP-1)、干扰素-1(IFN-1)、TNF-α、IL-12p70含量。结果:重组小鼠GM-CSF和IL-4诱导培养小鼠骨髓细胞4d,加入脂多糖(LPS)继续培养2d,近90%细胞高表达DCs特征性标志物CD11c,高表达抗原提呈分子MHC-Ⅱ和共刺激分子CD86、CD80,具有典型的树突状形态学特征和分泌炎症性细胞因子IL-6、IL-10、MCP-1、TNF-α和IL-12p70的功能。结论:小鼠骨髓细胞以重组小鼠GM-CSF和IL-4体外诱导培养的DCs具有较高的纯度,保持了体内的形态学、免疫表型及功能特征。

英文摘要：

Objective:To establish the method for cultivation and amplification of bone marrow-derived dendritic cells (DCs ) of mice in vitro, in order to provide an experimental base for the DC-targeting therapeutic study.Methods:Bone marrow cells of C57BL/6 mice were obtained and cultured in RPMI1640 containing recombi-nant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF)400 U/ml,interleukin-4 (IL-4)200 U/ml,and 10% fetal bovine serum invitro. On the 4th day,lipopolysaccharide (LPS)was added.Two days later, the morphology and ultrastucture of DCs were observed by phase contrast microscope and transmission electron microscope. The purity and immunophenotype of DCs were verified by flow cytometry on day 6. The contents of cytokines including IL-6,IL-10,monocyte chemotactic protein-1 (MCP-1),interferon-1 (IFN-1),tumor necrosis factor-α(TNF-α),IL-12p70 in supernatant were determined by cytometric bead array.Results:After cultured for 4 days followed by stimulation with LPS for 2 days,nearly 90% of mouse bone marrow cells expressed the charac-teristic DC marker-CD11c,with high level of antigen presentation molecule-major histocompability complex-Ⅱ(MHC-Ⅱ)and costimulatory molecules-CD86 and CD80. The cells possessed the typical morphologic feature of DCs and the function of secreting pro-inflammatory cytokines such as IL-6,IL-10,MCP-1,TNF-αand IL-12p70. Conclusions:Culturing invitro with GM-CSF and IL-4,bone marrow cells of mice can be induced to become DCs with high purity,with their characteristics of morphology,immunophenotype,and immunological functions.