增生性瘢痕成纤维细胞中分泌型凋亡相关蛋白1相互作用蛋白的研究

目的 寻找与分泌型凋亡相关蛋白1 （secreted apoptosis-related protein1,SARP1）存在相互作用的蛋白质分子,分析其分子机制.方法 成功构建的重组SARP1腺病毒载体Ad-SARP1,转染进入原代培养的增生性瘢痕成纤维细胞（HSFb）.免疫共沉淀法沉淀出蛋白,经聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后,用考马斯亮蓝染色,对SDS-PAGE胶上切取的电泳蛋白条带进行酶解与质谱分析,根据质谱分析获得的肽序列谱图自动进行数据库搜索.结果 在阳细胞对照组和导入Ad-SARP1感染的细胞当中,均共沉淀出7条蛋白带,相对分子质量分别约为93×103、43×103、40×103、37×103、31×103、26×103和12×103;在未加SARP1抗体组则没有沉淀出蛋白条带.分析蛋白条带得到6个可能与SARP1有相互作用的蛋白,分别为骨膜蛋白前体（periostin precursor或OSF-2）、牙周韧带相关蛋白1（asporin precursor或PLAP1）、磷酸甘油激酶1（phosphoglycerate kinase 1）、未知蛋白rCG50690、载脂蛋白（apolipoprotein A-I,Apo-AI）、硫氧还蛋白1（thioredoxin 1,TRX1）.结论 通过免疫共沉淀法结合液相色谱/质谱联用离子阱检测技术,可以获得SARP1的相互作用蛋白,为研究SARP1调控HSFb凋亡信号通路的作用机制提供了新的线索.

Interaction of SARP1 proteins in fibroblasts of hypertrophic scar

Objective To study interaction proteins with secreted apoptosis-related protein 1 （SARP1） in fibroblasts of hypertrophic scar and to analyze its molecular mechanisms.Methods The recombinant adenovirus Ad-SARP1 was successfully constructed and transfected into the fibroblasts of hypertrophic scar in culture.The proteins were precipitated by immunoprecipitation and separated by SDS-PAGE,then it was stained with Coomassie blue and proteins from SDS-PAGE gel electrophoresis strip were analyzed with enzymolysis and mass spectrometric in turn.The peptide sequences were obtained according to mass spectrometry and the database were searched automatically.Results The results showed that in control cells,Ad-SARP1 and Ad-EGFP infected cells,were precipitated 7 protein bands,their molecular weights were about 93 × 103,43 × 103,40 × 103,37 × 103,31 × 103,26 × 103 and 12× 103,respectively; without SARP1 antibody the protein bands did not precipitate.Analysis of the 6 protein bands showed that proteins that might interact with SARP1 included periostin precursor （OSF-2）,asporin precursor （PLAP1）,phosphoglycerate kinase 1,rCG50690,apolipoprotein A-I precursor （Apo-AI）,and thioredoxin 1 （TRX1）.Conclusions The interaction proteins of SARP1 can be obtained by immunoprecipitation combined with liquid chromatography/mass spectrometry and ion trap detection technology.These results provide new clues for the mechanism of SARP1 regulates the apoptosis signal pathway of HSFb.