期刊文献+

小鼠胚胎成纤维细胞条件培养液的蛋白质组学初步分析 被引量:1

Preliminary proteome analysis of mouse embryonic fibroblast conditioned medium
下载PDF
导出
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
  • 引文网络
  • 相关文献

参考文献17

  • 1Thomson JA, hskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[ J]. Science, 1998, 282(5391) : 1145-1147. 被引量:1
  • 2林戈,谢常青,卢光琇.人类胚胎干细胞体外分离与培养的初步研究[J].湖南医科大学学报,2002,27(5):484-484. 被引量:1
  • 3Reubinoff BE, Pera MF, Fong CY, et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro [ J ].Nat Biotech, 2000, 18(4) : 399-404. 被引量:1
  • 4Xu C, Inokuma MS, Denham J, et al. Freeder-free growth of undifferentiated human embryonic stem cells[ J ]. Nat Biotech, 2001,19(10) : 971-974. 被引量:1
  • 5Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells [ J ]. Proteomics, 2002, 2 ( 9 ) :1187-1203. 被引量:1
  • 6Xie C, Lin G, Lu G. Priliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro [ J ].Chin Sci Bull, 2002, 47(24) : 1880-1882. 被引量:1
  • 7Xie CQ, Lin G, Luo KL, et al. Newly expressed proteins of mouse embryonic fibroblasts irradiated to be inactive [ J ]. Biochem Biophys Res Commun, 2004, 315(3) : 581-588. 被引量:1
  • 8Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[ J ]. Anal Biochem, 1976, 72 : 248-254. 被引量:1
  • 9Gharahdaghi F, Weinberg CR, Meagher DA, et al. Mass spectrometric of protein from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity [ J ].Electrophoresis, 1999, 20 ( 3 ) : 601-605. 被引量:1
  • 10Yuan X, Russell T, Wood G, et al. Analysis of the human lumbar cerebrospinal fluid proteome [ J ]. Electrophoresis, 2002, 23 ( 7-8) : 1185-1196. 被引量:1

同被引文献14

  • 1刘永刚,李芳菲,陈地龙,王莎莉,王亚平.以STO细胞和hELF作为胚胎生殖细胞培养饲养层的比较[J].第四军医大学学报,2006,27(18):1704-1707. 被引量:7
  • 2Amit M, Shariki C, Margulets V, et al. Feeder layer-and serum free culture of human embryonic stem cells [J]. Biol Reprod, 2004, 70(3): 837-845. 被引量:1
  • 3Taupin P. Derivation of embryonic stem cells for cellular therapy: challenges and new strategies [J]. Med Sci Monit, 2006, 12(4) RA75-RA78. 被引量:1
  • 4Sidhu KS, Lie KH, Tuch BE. Transgcnic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection [J]. Stem Cells Dev, 2006, 15(5): 741-747. 被引量:1
  • 5Lee JB, Song JM, Lee JE, et al. Available human feeder cells for the maintenance of human embryonic stem cells [J]. Reproduction, 2004, 128(6): 727-735. 被引量:1
  • 6Amit M, Margulets V, Segev H, et al. Human feeder layers for human embryonic stem cells [J]. Biol Reprod, 2003, 68 (6): 2150-2156. 被引量:1
  • 7Xu C, Inokuma MS, Denham J, et al. Feeder free growth of un differentiated human embryonic stem cells [J]. Nat Bioteehnol, 2001, 19(10): 971-974. 被引量:1
  • 8Stojkovic P, Lako M, Przyborski S, et al. Human-serum matrix supports undifferentiated growth of human embryonic stem cells [J]. Stem Cells, 2005, 23(7): 895-902. 被引量:1
  • 9Davey RE, Onishi K, Mahdavi A, et al. LIF-mediated control of embryonic stem cell self-renewal emerges due to an autoregulatory loop [J]. FASEB J, 2007, 21(9) : 2020-2032. 被引量:1
  • 10Kueh J, Richards M, Ng SW, et al. The search for factors in human feeders that support the derivation and propagation of human embryonic stem cells: preliminary studies using transcriptome profiling by serial analysis of gene expression [J]. Fertil Steril, 2006, 85(6): 1843-1846. 被引量:1

引证文献1

二级引证文献1

;
使用帮助 返回顶部