CAJ | 학술논문

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.
목적 응용동위소표기상대화절대정량(iTRAQ)기술관찰핵인자-κB(NF-κB)필수분자결합적소분자다태(NBD)간예종류배사인자-α(TNF-α)자격하성골세포분화과정중단백질표체조적변화.방법 기원C2C12세포접충우골형태발생단백-2(BMP-2)유도분화체계중유도작위분화세포모형,실험분위3조:대조조(B조)즉분화세포모형미가기타자격,실험조(BT조)즉분화세포모형첨가TNF-α,간예조(BTP조)즉분화세포모형첨가TNF-α급NBD다태,공동부육분화7d후제취단백,이iTRAQ시제표기후진행질보검측병이연건분석차이표체적단백질.결과 TNF-α명현억제성골세포분화,이NBD다태가부분개선TNF-α대성골세포분화적억제.iTRAQ시제표기적B조여B+T조세포단백질표체보분석사선출명현차이표체단백질점위76개,표체상조적단백질점59개,하조적단백질점17개;BT조여BTP조세포단백질표체보분석사선출명현차이표체단백질점위43개,표체상조적단백질점25개,하조적단백질점18개.기중3조간성골세포특이인자(Postn)、ATP매개리자운수인자(Atp2a3)、SR의뢰단백CTD관련인자-1(Scafl)、기동단백-F교련단백(Actn2)、ATP합성매,경리자전운,선립체복합체-F1,Delta아기(Atp5d)、연신체을선전이매복합체아기-2(Elp2)、Atp5]、C1형니만-필극질병(Npc1)등8개단백표체출현동태변화,제시상술단백가능여염증자격성골분화궤제유관,NBD다태지외상유기타약물파점간예염증대성골분화적억제.결론 iTRAQ기술시연구세포분자단백개변적유효적단백질조학방법.Postn、Atp2a3、Scaf1、Actn2、Atp5d、Elp2、Atp5l급Npc1가능작위염증자격성골분화궤제연구적후선파표.
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.