CAJ | 학술논문

目的应用同位素标记的绝对和相对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术筛选并鉴定不同预后的胆道闭锁患儿肝脏中表达的差异蛋白质,并进行生物信息学分析.方法收集Ⅲ型胆道闭锁患儿Kassi手术时肝脏组织,术后对患儿预后进行随访,根据3个月时黄疸是否消退和自体肝存活时间,将标本筛选并分为两组,预后较好组(术后3个月时血清总胆红素≤25 μmol/L,自体肝存活时间≥2年)和预后较差组(术后3个月时血清总胆红素＞25μmol/L,自体肝存活时间＜1年),每组患儿标本各10例.将样本经iTRAQ标记和液相色谱分离后,用质谱仪进行鉴定和相对定量分析,对获得的差异蛋白进行PANTHER分类和生物功能分析,并对差异表达的蛋白Mx1进行了Western-blot验证.结果经质谱鉴定共获得蛋白5 177个,其中差异表达倍数大于1.5倍的蛋白共323种,包括在预后较好组上调的蛋白131种和在预后较差组上调的蛋白192种.经PANTHER蛋白分类和功能分析,差异表达的蛋白主要涉及代谢过程、免疫过程等14个生物过程.其中在预后较好组差异表达倍数前10位的上调蛋白,几乎全部参与了病毒感染后的免疫应答过程.结论 iTRAQ技术能够快速、有效地进行肝脏差异蛋白质组学的研究,能筛选出多种与胆道闭锁病因和预后相关的蛋白,为进一步研究胆道闭锁发病机制提供了实验依据.
목적 응용동위소표기적절대화상대정량(isobaric tags for relative and absolute quantitation,iTRAQ)기술사선병감정불동예후적담도폐쇄환인간장중표체적차이단백질,병진행생물신식학분석.방법 수집Ⅲ형담도폐쇄환인Kassi수술시간장조직,술후대환인예후진행수방,근거3개월시황달시부소퇴화자체간존활시간,장표본사선병분위량조,예후교호조(술후3개월시혈청총담홍소≤25 μmol/L,자체간존활시간≥2년)화예후교차조(술후3개월시혈청총담홍소＞25μmol/L,자체간존활시간＜1년),매조환인표본각10례.장양본경iTRAQ표기화액상색보분리후,용질보의진행감정화상대정량분석,대획득적차이단백진행PANTHER분류화생물공능분석,병대차이표체적단백Mx1진행료Western-blot험증.결과 경질보감정공획득단백5 177개,기중차이표체배수대우1.5배적단백공323충,포괄재예후교호조상조적단백131충화재예후교차조상조적단백192충.경PANTHER단백분류화공능분석,차이표체적단백주요섭급대사과정、면역과정등14개생물과정.기중재예후교호조차이표체배수전10위적상조단백,궤호전부삼여료병독감염후적면역응답과정.결론 iTRAQ기술능구쾌속、유효지진행간장차이단백질조학적연구,능사선출다충여담도폐쇄병인화예후상관적단백,위진일보연구담도폐쇄발병궤제제공료실험의거.
Objective To identify and quantify total proteins in liver tissue of patients with biliary atresia (BA) by isobaric tags for relative and absolute quantification (iTRAQ) technology to decipher the relationship between significant protein and its prognosis.Methods According to the serum level of total bilirubin (TBIL) in 3 months and native liver survival time (NLST),the patients were divided into 2 groups of good prognosis (3M TBIL≤ 25 μmol/L,NLST＞2 years) and poor prognosis (3M TBIL＞25μmol/L,NLST＜ 1 year) (n =10 each).Liver tissues were collected during Kasai operation.And iTRAQ technique was used to identify and quantify the expression of differential proteins.Then differential proteins were classified by Panther classification system.The results were further validated by Western blot.Results A total of 5177 proteins were identified by mass spectrometry.Among them,323 differential proteins with expression folds of over 1.5 were identified.Among these proteins,as compared with poor prognosis group,131 proteins were up-regulated and 192 proteins down-regulated in good prognosis group.These differential proteins were related with 14 kinds of biological processes (e.g.metabolic and immune system).Conclusions The technique of iTRAQ is effective for comparative proteomic studies.Thus these identified differential proteins will provide further clues for studying the pathogenesis and prognosis of BA.