以iTRAQ技术分析大鼠肝纤维化发展成肝癌过程中肝组织差异表达的蛋白质

Proteomic identification of differentially expressed proteins in rat hepatocarcinogenesis using iTRAQ technology

目的:分析大鼠肝纤维化发展成为肝癌过程中肝组织差异表达的蛋白,以寻找其发展过程中的关键蛋白.方法:以二乙基亚硝胺(diethylinitrosamine,DEN)建立大鼠肝纤维化-肝癌模型,采用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iT RAQ)技术结合2D-LC-MS/MS(two dimensional liquid chromatography-tandem mass spectrometry)定量蛋白质组学技术,分析和鉴定建模过程中的第4、10、12、18周肝组织蛋白质的差异表达,经生物信息学分析差异表达的蛋白质功能.结果:质谱分析共鉴定到530种蛋白质.4个时间点鉴定到的差异蛋白质数量分别为128、113、106、127个,均重复鉴定到的差异蛋白为49个,其中上调蛋白37个,下调蛋白12个.通过对这些差异蛋白进行功能分析,发现他们参与翻译后修饰、转录、重组和信号传导等过程.结论:iTRAQ结合LC-ESI-MS/MS定量蛋白质组学技术能整体、动态分析的大鼠肝纤维化演变肝癌过程中的蛋白质表达变化,其中鉴定到过氧化物还原酶1(peroxiredoxin-1,Prdx1)、过氧化物还原酶2(peroxiredoxin-2,Prdx2)、硫代硫酸硫基转移酶(thiosulfate sulfurtransferase,Tst)、真核翻译起始因子3b(eukaryotic translation initiation factor 3subunit b,e IF3b)、α2-HS-糖蛋白(alpha-2-HS-glycoprotein,Ahsg)和超氧化物歧化酶1(superoxide dismutase 1,SOD1)蛋白可能是肝纤维化发展为肝癌过程中的关键蛋白,值得进一步深入探讨研究.

AIM： To identify differentially expressed proteins in rat hepatocarcinogenesis. METHODS： Sprague-Dawley rats were randomly divided into a diethylinitrosamine （DEN） group and a normal control group. The DEN group was administrated 0.01% DEN solution in water for 18 wk, and the control group was given vehicle. The animals were sacrificed at the end of the 4~, 10th, 12th, or 18th week. Liver tissue proteins were quantified and identified using the isobaric tags for relative an absolute quantitation （iTRAQ） technology with two dimensional liquid chromatography- tandem mass spectrometry （2DLC-MS/MS）. The functions of differentially expressed proteins were analyzed by bioinformatics. RESULTS： A total of 530 proteins were identified by MS, and the numbers of differentially expressed proteins at the four time points were 128, 113, 106 and 127, respectively. Forty-nine proteins were identified simultaneously at two or more time points, of which 37 were up-regulated and 12 down-regulated. Bioinformatics analysis indicated that differentially expressed proteins are associated with post-translational modification, transcription, recombination and signal transduction pathways.CONCLUSION： The protein changes in the process of rat hepatocarcinogenesis can be observed dynamically by iTRAQ combined with LC-ES｜-MS/MS. Six proteins （peroxiredoxin-1, peroxiredoxin-2, thiosulfate sulfurtransferase, eukaryotic translation initiation factor 3 subunit b, alpha-2-HS-glycoprotein, and superoxide dismutase 1） are associated closely with hepatic fibrosis or hepatic carcinoma, and they may play critical roles in hepatocarcinogenesis.