非酒精性脂肪性肝病患者的肝组织差异蛋白的定量分析:基于iTRAQ技术

目的 应用蛋白质组学在非酒精性脂肪性肝炎（NASH）肝脏组织中筛选差异蛋白,寻找关键的作用靶点。方法 收集符合纳入标准的肝脏组织，通过 HE 染色病理切片筛选出非酒精性脂肪性肝炎样本（NASH组）3例和正常对照样本3例。提取NASH组及正常对照组的肝脏蛋白，使用iTRAQ试剂对多肽进行标记进行液相色谱串联质谱（LC-MS/MS）检测，用蛋白质鉴定软件Mascot2.3.02比对UniProt蛋白数据库搜索鉴定，应用GO数据库对差异表达蛋白质进行注释和富集，应用KEGG数据库进行差异蛋白质涉及信号通路富集。实时荧光定量PCR（qPCR）检测显著差异表达蛋白对应的mRNA表达水平。结果 NASH组和对照组相比，以差异倍数（>1.2或<0.8）且P<0.05为阈值质谱分析鉴定到648个显著差异蛋白，其中表达量上调的蛋白有246种，下调的蛋白有402种。GO功能富集分析结果显示，差异表达蛋白主要参与小分子代谢、有机酸代谢、含氧酸代谢等生物学过程，在代谢途径、补体凝血级联、核糖体等KEGG通路上富集。荧光定量PCR对差异倍数（>2.0或<0.5）且P<0.05的25个显著差异表达蛋白进行筛选，共有6个蛋白与蛋白组学的结果趋势一致，包括5个下调蛋白：Jumonji 蛋白（JARID2）、莱伯西林样蛋白（LCA5L）、突触素1（SYN1）及胶原α-1（XIII）链（COL13A1）、FYVE，RhoGEF和PH结构域蛋白5（FGD5），以及1个上调蛋白：谷胱甘肽S-转移酶Mu 4（GSTM4）。结论 iTRAQ技术和生物信息学方法在NASH肝脏组织筛选出差异表达蛋白648个，其中JARID2、SYN1、COL13A1、FGD5、GSTM4可能是NASH的关键靶向蛋白。

Quantitative analysis of differential proteins in liver tissues of patients with non-alcoholic steatohepatitis using iTRAQ technology

Objective To screen differentially expressed proteins (DSPs) in the liver tissues of patients with nonalcoholic steatohepatitis (NASH) using proteomic technologies to identify potential therapeutic targets of NASH. Methods Liver tissue specimens were obtained from 3 patients with pathologically confirmed NASH and 3 normal control subjects. The total proteins were extracted from the specimens, and iTRAQ reagent was used to label the peptides for liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. The DSPs were identified by comparing the data against UniProt protein database using Mascot2.3.02 software and were annotated and enriched using GO database; KEGG database was used for enrichment of the pathways involving these proteins. Real-time fluorescent quantitative PCR (qPCR) was performed to detect the mRNA expressions of the significant DSPs in NASH. Results By the criteria that a DSP has >1.2 or <0.8 fold difference between NASH group and the control group and with P<0.05 as the threshold, a total of 648 significant DSPs in NASH were identified, including 246 up-regulated and 402 down-regulated proteins. GO functional enrichment analysis showed that the DSPs were involved mainly in small molecule metabolism, organic acid metabolism, oxygen acid metabolism and other biological processes, and were enriched in KEGG pathways including the metabolic pathways, complement coagulation cascades, and ribosomes. Among the 25 DEPs with a fold difference >2.0 or <0.5 (P<0.05), 6 proteins showed consistent results between qPCR verification and proteomic analysis, including 5 down-regulated proteins: Jumonji protein (JARID2), Lebasillin-like protein (LCA5L), synaptophysin 1 (SYN1) and collagen α-1 (XIII) chain (COL13A1), FYVE, RhoGEF and PH domain protein 5 (FGD5), and 1 upregulated protein glutathione S-transferase Mu 4 (GSTM4). Conclusion We identified 648 DEPs in the liver tissue of patients NASH using iTRAQ technology and bioinformatics methods, and among them JARID2, SYN1, COL13A1, FGD5, and GSTM4 may serve as the key target proteins of NASH.