小鼠骨髓来源未成熟树突状细胞蛋白表达的分析

Protein expression analysis of immature mouse bone marrow-derived dendritic cells

目的利用双向电泳和质谱技术对小鼠骨髓来源未成熟树突状细胞(dendritic cells,DC)表达的蛋白质进行分析。方法 IL-4和GM-CSF诱导未成熟DC的分化,提取细胞总蛋白,定量。双向凝胶电泳分离蛋白质组分,胶体银染显色,PDQuest进行图像分析后选取蛋白点,胶内酶解后MALDI-TOF MS进行肽指纹图谱鉴定,MS-Fit分析质谱数据。结果细胞生长情况和表面标志物检测表明获得了高纯度的未成熟DC。图像分析结果显示,DC中检测到了(660±10)个蛋白点,主要分布在相对分子质量28×103～100×103,等电点5.0～9.0之间。鉴定了87个蛋白点,其中与未成熟DC免疫调节功能相关的蛋白质有40个(45.98%);与大分子代谢相关的有37个(42.53%)。结论成功分离到了约660个小鼠骨髓来源未成熟DC表达的蛋白质,鉴定出了其中87个蛋白。

Objective To analyze the proteins of immature mouse bone marrow-derived dendritic cells（BMDCs） by 2-dimentional electrophoresis（2-DE） and peptide mass fingerprinting.Methods Interleukin-4（IL-4） and granulocyte-macrophage colony-stimulating factor（GM-CSF） were employed to induce differentiation of mouse bone marrow cells into immature mouse BMDCs.Proteins were extracted from the DCs,separated by 2-DE,stained by improved silver method,analyzed by PDQuest software,and identified by peptide mass fingerprinting and MS-Fit.Results High-purity immature mouse BMDCs were obtained,as confirmed by detecting cell growth condition and surface markers.Image analysis result showed that 660±10 protein spots were separated and mainly distributed in a relative molecular weight range of（28-100）×103 and an isoelectric point range of 5.0-9.0.Among the 87 identified spots,40（45.98%） were related to immunoregulation function of immature DCs and 37（42.53%） to macromolecular metabolism.Conclusion Six hundred and sixty proteins are separated from immature mouse BMDCs,and 87 proteins are identified.