树突状细胞体外培养及其诱导抗肿瘤免疫作用.doc

树突状细胞体外培养及其诱导抗肿瘤免疫作用 　作者：张建芳，于文彬，徐修礼，苏明权，马越云，刘家云，丁振若 【关键词】 树突状细胞 　　关键词: 树突状细胞;LAK细胞;免疫治疗 　　摘 要:目的 建立体外培养扩增树突状细胞（DC）的方法，并观察其诱导的抗肿瘤免疫作用. 方法 分离外周血单个核细胞，应用GM-CSF及IL-4诱导DC产生，用TNF-α和PGE2 促进其成熟，并用肺癌细胞系GLC-82可溶性抗原致敏.将成熟DC与自体LAK细胞混合培养（DC-LAK），用MTT法检测DC-LAK细胞及LAK细胞对多种肿瘤细胞系的杀伤作用. 结果 经GM-CSF，IL-4，TNF-α，PGE2 作用后，细胞表达CD1 a阳性率为71.0%，CD83 50.0%，CD14 阳性率低于10.0%，高表达HLA-Ⅰ，HLA-Ⅱ和CD45 ，为典型的DC表型特征.DC-LAK和LAK细胞对4种肿瘤细胞（GLC-82，A549，moser和MCF-7）的杀伤率，前者为84.7%，66.9%，49.3%和56.0%，后者为43.5%，31.3%，45.0%和32.4%. 结论 成功的诱导出具有典型形态特征及增强LAK细胞杀伤活性的DC. 　　In vitro generation of human blood dendritic cells for active tumor immunotherapy 　　Abstract:AIM To establish a method to culture dendritic cells（DC）in vitro and to observe the anti-tumor effect in-duced by DC.METHODS Dendritic cells were derived from PBMC stimulated with GM-CSF and IL-4and DC were pulsed with tumor soluble antigen abstracted from lung can-cer cell line GLC-82.TNF-αand PGE2 were used to enhance antigen presenting ability of DC.Phenotype of DC were ana-lyzed through FACS.Mature DC were cocultured with LAK cells and the mixed cells were named DC-LAK.Cytotoxicity of DC-LAK and LAK were measured by MTT assay.RESULTS The cultured DC expressed HLA-Ⅰ，HLA-Ⅱand CD45 and positive rates of CD1 a，CD83 and CD14 were71.0%，50.0%and less than10.0%respectively.Killing rates of DC-LAK and LAK to the four cell lines（GLC-82，A549，moser and MCF-7）were84.7%，66.9%，49.3%，56.0%and43.5%，31.3%，45.0%，32.4%respectively.CONCLUSION DC that have typical phenotype and could be enhance cytotoxity of LAK could be induced from PBMC suc-cessfully. 　　0 引言 　　近年来树突状细胞（DC）可有效地递呈可溶性肿瘤抗原，增强机体对肿瘤的特异性免疫应答，并在临床取得了成绩［1，2］ .我们将外周血中贴壁的单个核细胞作为DC的前体细胞，联合应用一组细胞因子诱导获得了纯度较高的人树突状细胞，并将之与LAK细胞结合，体外诱导产生了相对高效而特异的抗肿瘤免疫作用. 　　1 材料和方法 　　1.1 材料 　　基因重组人IL-4，TNF-α和GM-CSF为Promega公司产品，PGE2 购自Sigma公司，淋巴细胞分离液购自上海医学试剂厂，IL-2为本校基因研究所产品，抗CD1 a-FITC和抗CD83 -FITC购自晶美公司，HLA-Ⅰ，HLA-Ⅱ抗体及羊抗鼠-FITC购自北京邦定公司，酶联免疫检测仪为Labsystem产品;CD14 /CD45 双色荧光标记抗体由西京医院免疫科提供.肺癌A549为本校基础部免疫学教研室提供;乳腺癌MCF-7细胞引自军事医学科学院;大肠癌moser细胞、肺癌G