摘要
目的:运用双向电泳、质谱技术对小鼠胚胎成纤维细胞饲养层条件培养液进行蛋白质组学分析,寻找 其中可能具有抑制分化作用的蛋白因子。方法:用60Coγ 射线照射昆明白小鼠胚胎成纤维细胞,制成饲养层,置于 ITS(胰岛素 转铁蛋白 硒钠盐)培养液中培养24h,收集条件培养液。用冷冻干燥、凝胶过滤层析、冷丙酮沉淀的方 法从条件培养液中提取蛋白质样品,经双向电泳和银染得到双向电泳图谱,用Imagemasterelite2.0软件进行图像 分析。取蛋白点进行酶解,再用CapLC ESI Q TOF质谱进行分析,使用Mascot软件在NCBInr数据库中进行搜索。 结果:双向电泳得到(221±67)个蛋白点的银染图谱,初步分析鉴定13个蛋白点。结论:昆明白小鼠胚胎成纤维细 胞条件培养液中含有β 半乳糖苷结合蛋白、半胱氨酸富集的酸性分泌蛋白(SPARC)等成分。
Objective To perform the proteome analysis of conditioned medium prepared from mouse embryonic fibroblast feeder layers by 2-dimensional (2D) electrophoresis and mass spectrometry and to find out the possible differentiation-inhibitory factor in conditioned medium. Methods Feeder layers were prepared by 60Co γ-irradiation on mouse embryonic fibroblast. Insulin-transferrin-sodium selenite supplemented medium was used to culture the feeder layers for 24 hours. The condioned medium prepared from mouse embryonic fibroblast feeder layers were made into powder by lyophilization, the re-dissolved solution was applied to Sephadex G-50 gel filtration chromatography, and then cold acetone was used to precipitate the proteins in the eluted solution. The protein samples were applied to 2D electrophoresis. The 2D images were analyzed by 2D image analysis software. Selected protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCBInr batabase using Mascot MS/MS Ions Search. Results The protein samples extracted from mouse embryonic fibroblast feeder layersconditioned medium could be used for 2D electrophoresis. On 2D images, there were (221±67) spots. Most of the proteins were located in the region of MW 20~70 kD,pI 4~8. Using mass spectrometry, we preliminarily identified 13 spots: 3 keratins, 3 transferrins, 1 trypsin precursor, 2 unknown proteins (3 spots), 1 connexin 46, 1 beta-galactoside binding protein, and 1 secreted protein, acidic and rich in cysteine. Conclusion Conditioned medium prepared from mouse embryonic fibroblast feeder layers contain beta-galactoside binding protein and secreted protein, acidic and rich in cysteine.
出处
《中南大学学报(医学版)》
CAS
CSCD
北大核心
2005年第1期11-15,共5页
Journal of Central South University :Medical Science
基金
国家自然科学基金(30300179)
国家自然科学基金重点项目(30030070)
国家973重大基础研究项目(2001cb510103)
关键词
小鼠
胚胎成纤维细胞
饲养层
条件培养液
双向电泳
质谱技术
干细胞分化抑制因子
mouse embryonic fibroblast
feeder layers
conditioned medium
two-dimensional electrophoresis
mass spectrometry
stem cell differentiation-inhibitory factor