摘要
目的应用双向凝胶电泳-质谱法分析正常人及类风湿关节炎（rheumatoid arthritis，RA）患者滑膜成纤维细胞蛋白质组表达差异，探讨差异蛋白在类风湿关节炎发病中的作用。方法采用固相pH梯度（immobilized pH gradient，IPG）双向凝胶电泳（two—dimensional gel eleetrophoresis，2-DE）分离正常人及类风湿关节炎患者滑膜成纤维细胞总蛋白质，银染显色，PDQuest软件分析图像，比较两种细胞蛋白质组表达差异，对差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行鉴定，并用Western blot验证部分表达差异蛋白质。结果①采用窄范围的pH4～7IPG胶条进行2-DE分析，正常人及RA患者滑膜细胞蛋白胶图上分别平均显示852、837个点。有49个蛋白点在正常、RA滑膜细胞问有2倍以上的显著性量变。②分别在胶图上共选取40个表达水平存在明显差异的蛋白质点进行质谱鉴定，鉴定出23个在RA患者滑膜细胞中表达上调蛋白质。Western blot验证Enolase α、Annexin I、CathepsinD、SOD2、Peroxiredoxin2在RA滑膜细胞中表达显著升高。结论正常人及类风湿关节炎患者滑膜成纤维细胞表达蛋白质组存在差异。这些差异蛋白可能参与类风湿关节炎的发病。
Objective To understand the possible role of some proteins expressed by human synovial fibroblasts （SFs） in the pathogenesis of rheumatoid arthritis. Methods The expression difference of synovial fibroblast proteins between rheumatoid arthritis （RA） patients and healthy controls was analyzed by 2-DE. The differential expression spots were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry （MALDI-TOF-MS）, followed by bioinformatics analysis, some of which were validated by Western blot. Results Using 12% SDS-PAGE following pH 4 -7 IPG strips in IEF, averagely 837 and 852 protein spots were detected in RA patients and normal subjects, respectively. Gel image analysis revealed that there were 49 differential protein spots. By peptide mass fingerprinting strategy, we identified 40 protein spots derived from gels of SFs in RA patients among differential spots and 23 valid proteins were obtained. Western blot analysis showed that expressions of Enolase or, Annexin I, Cathepsin D, SOD2, Peroxiredoxin 2 were significmtly higher in SFs from RA patients than those from normal subjects, which was consistent with proteome analysis. Conclusion The differential proteins might be involved in inflammation of synovitis in RA.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第2期166-169,共4页
Journal of Third Military Medical University
关键词
滑膜成纤维细胞
蛋白质组
双向电泳
类风湿关节炎
synovial fibroblasts
proteome
two-dimensional electrophoresis
rheumatoid arthritis
