小鼠骨髓来源未成熟树突状细胞蛋白表达的分析.docVIP

小鼠骨髓来源未成熟树突状细胞蛋白表达的分析 易发平，焦庆昉，符少月，卜友泉，刘革力，宋方洲（重庆 400016，重庆医科大学基础医学院生物化学与分子生物学教研室，分子医学与肿瘤研究中心） [摘要]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。 [关键词] 树突状细胞；蛋白质组；双向电泳；肽指纹质谱 [中图法分类号] [文献标志码]A The analysis of protein expression in mouse myeloid dendritic cells Yi Faping，Jiao Qingfang，Fu Shaoyue，Bo Youquan，Liu Geli，Song Fangzhou（Department of Biochemistry and Molecular Biology， Molecular Medicine and Cancer Research Center；Chongqing Medical University，Chongqing 40016，China） [Abstract] Objective To afford experimental proof for application of dendritic cells （DC）， the protein， expressed in immature DC derived from mouse myeloid， were separated by 2-dimention electrophoresis （2-DE）， and identified by peptide mass fingerprinting method. Methods The differentiation of immature DC were induced by interleukin-4 （IL-4） and granulocyte-macrophage colony-stimulating factor （GM-CSF）. The proteins were extracted from the DC， separated by 2-DE， stained by improved silver method， analyzed by PDQuest software， and identified by peptide mass fingerprinting method and MS-Fit. Results The high pure immature DC were obtained through detecting Cell growth and surface markers. 660 ± 10 protein spots were successfully separated. Most of the molecular weight of protein were from 28 ×103 to 100 ×103， and isoelectric point from 5.0 to 9.0. 87 spots were analyzed. A total of 40 proteins （45.98%） were related to immune functions and 37 proteins （42.53%） related to macromolecules metabolism in DC. Conclusion 660 protein， expressed in immature DC derived from mouse myeloid， were separated successfully and 87 spots were analyzed. [Key words]d