基于同位素标记相对和绝对定量技术核因子-KB必需分子结合的小分子多肽改善肿瘤坏死因子-α抑制成骨细胞分化的蛋白质组学研究

Isobaric tags for relative and absolute quantitation-based quantitative protein expression profiling of NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha

目的 应用同位素标记相对和绝对定量（iTRAQ）技术观察核因子-κB（NF-κB）必需分子结合的小分子多肽（NBD）干预肿瘤坏死因子-α（TNF-α）刺激下成骨细胞分化过程中蛋白质表达组的变化.方法 肌原C2C12细胞接种于骨形态发生蛋白-2（BMP-2）诱导分化体系中诱导作为分化细胞模型,实验分为3组：对照组（B组）即分化细胞模型未加其他刺激,实验组（BT组）即分化细胞模型添加TNF-α,干预组（BTP组）即分化细胞模型添加TNF-α及NBD多肽,共同孵育分化7d后提取蛋白,以iTRAQ试剂标记后进行质谱检测并以软件分析差异表达的蛋白质.结果 TNF-α明显抑制成骨细胞分化,而NBD多肽可部分改善TNF-α对成骨细胞分化的抑制.iTRAQ试剂标记的B组与B＋T组细胞蛋白质表达谱分析筛选出明显差异表达蛋白质点为76个,表达上调的蛋白质点59个,下调的蛋白质点17个;BT组与BTP组细胞蛋白质表达谱分析筛选出明显差异表达蛋白质点为43个,表达上调的蛋白质点25个,下调的蛋白质点18个.其中3组间成骨细胞特异因子（Postn）、ATP酶钙离子运输因子（Atp2a3）、SR依赖蛋白CTD关联因子-1（Scafl）、肌动蛋白-F交联蛋白（Actn2）、ATP合成酶,氢离子转运,线粒体复合体-F1,Delta亚基（Atp5d）、延伸体乙酰转移酶复合体亚基-2（Elp2）、Atp5]、C1型尼曼-匹克疾病（Npc1）等8个蛋白表达出现动态变化,提示上述蛋白可能与炎症刺激成骨分化机制有关,NBD多肽之外尚有其他药物靶点干预炎症对成骨分化的抑制.结论 iTRAQ技术是研究细胞分子蛋白改变的有效的蛋白质组学方法.Postn、Atp2a3、Scaf1、Actn2、Atp5d、Elp2、Atp5l及Npc1可能作为炎症刺激成骨分化机制研究的候选靶标.

Objective To screen for the differentially expressed proteins of NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha.Methods The myoblast C2C12 cells as cellular model were cultured with serum-free DMEM and divided into three groups cultured with different stimulus.Cells were induced with bone morphogenetic protein （BMP-2） as the control group （group B）,cells were induced with BMP-2 and treated with BMP-2 and tumor necrosis factor-α （TNF-α） as the experimental group （group BT）,cells were induced with BMP-2 and treated with BMP-2,TNF-α and NBD peptide as the treatment group （group BTP）.The total protein at 7 d during the differentiation was extracted and analyzed by isobaric tags for relative and absolute quantitation （iTRAQ） technology coupled with mass spectrometric analysis.Results A total of 76 significant protein spots were found by mass spectrometric analysis,among which,59 spots were up-regulated and 17 spots were down-regulated between group B and group BT.And 43 significant protein spots were found by mass spectrometric analysis,among which,25 spots were up-regulated and 18 spots were down-regulated between group BT and group BTP.Among three groups,protein expression of periostin protein （Postn）,sarcoplasmic reticulum Ca observed in the ATP share 2 ＋ enzyme （Atp2a3）,SR dependent protein （Scaf1）,alpha-actinin 2 （Actn2）,ATP synthase subunit D （Atp5d）,5 extended protein 2 （Elp2）,Atp5l and Niemann-Pick type C1 （Npc1） were dynamic changed,implying a close linkage between above protein and osteoblast differentiation impaired by inflammation,there are other drug target to promotes osteoblast differentiation inhibited by inflammation.Conclusion iTRAQ is a useful technologic method in proteomic study of cell differentiation.Postn,Atp2a3,Scaf1,Actn2,Atp5d,Elp2,Atp5l,Npc1 could serve as potential target molecules for the mechanism study on steoblast differentiation inhibited by inflammation.