中华医学美学美容杂志
中華醫學美學美容雜誌
중화의학미학미용잡지
CHINESE JOURNAL OF MEDICAL AESTHETICS AND COSMETOLOGY
2014年
5期
381-384
,共4页
王儆%王雪梅%王珍祥%陈亮%李世荣
王儆%王雪梅%王珍祥%陳亮%李世榮
왕경%왕설매%왕진상%진량%리세영
分泌型凋亡相关蛋白1%增生性瘢痕%成纤维细胞%免疫共沉淀%相互作用蛋白
分泌型凋亡相關蛋白1%增生性瘢痕%成纖維細胞%免疫共沉澱%相互作用蛋白
분비형조망상관단백1%증생성반흔%성섬유세포%면역공침정%상호작용단백
Secreted apoptosis-related protein 1 (SARP1)%Hypertrophic scar%Fibroblast%Immunoprecipitation%Interaction protein
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.
目的 尋找與分泌型凋亡相關蛋白1 (secreted apoptosis-related protein1,SARP1)存在相互作用的蛋白質分子,分析其分子機製.方法 成功構建的重組SARP1腺病毒載體Ad-SARP1,轉染進入原代培養的增生性瘢痕成纖維細胞(HSFb).免疫共沉澱法沉澱齣蛋白,經聚丙烯酰胺凝膠電泳(SDS-PAGE)分離後,用攷馬斯亮藍染色,對SDS-PAGE膠上切取的電泳蛋白條帶進行酶解與質譜分析,根據質譜分析穫得的肽序列譜圖自動進行數據庫搜索.結果 在暘細胞對照組和導入Ad-SARP1感染的細胞噹中,均共沉澱齣7條蛋白帶,相對分子質量分彆約為93×103、43×103、40×103、37×103、31×103、26×103和12×103;在未加SARP1抗體組則沒有沉澱齣蛋白條帶.分析蛋白條帶得到6箇可能與SARP1有相互作用的蛋白,分彆為骨膜蛋白前體(periostin precursor或OSF-2)、牙週韌帶相關蛋白1(asporin precursor或PLAP1)、燐痠甘油激酶1(phosphoglycerate kinase 1)、未知蛋白rCG50690、載脂蛋白(apolipoprotein A-I,Apo-AI)、硫氧還蛋白1(thioredoxin 1,TRX1).結論 通過免疫共沉澱法結閤液相色譜/質譜聯用離子阱檢測技術,可以穫得SARP1的相互作用蛋白,為研究SARP1調控HSFb凋亡信號通路的作用機製提供瞭新的線索.
목적 심조여분비형조망상관단백1 (secreted apoptosis-related protein1,SARP1)존재상호작용적단백질분자,분석기분자궤제.방법 성공구건적중조SARP1선병독재체Ad-SARP1,전염진입원대배양적증생성반흔성섬유세포(HSFb).면역공침정법침정출단백,경취병희선알응효전영(SDS-PAGE)분리후,용고마사량람염색,대SDS-PAGE효상절취적전영단백조대진행매해여질보분석,근거질보분석획득적태서렬보도자동진행수거고수색.결과 재양세포대조조화도입Ad-SARP1감염적세포당중,균공침정출7조단백대,상대분자질량분별약위93×103、43×103、40×103、37×103、31×103、26×103화12×103;재미가SARP1항체조칙몰유침정출단백조대.분석단백조대득도6개가능여SARP1유상호작용적단백,분별위골막단백전체(periostin precursor혹OSF-2)、아주인대상관단백1(asporin precursor혹PLAP1)、린산감유격매1(phosphoglycerate kinase 1)、미지단백rCG50690、재지단백(apolipoprotein A-I,Apo-AI)、류양환단백1(thioredoxin 1,TRX1).결론 통과면역공침정법결합액상색보/질보련용리자정검측기술,가이획득SARP1적상호작용단백,위연구SARP1조공HSFb조망신호통로적작용궤제제공료신적선색.
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.
