应用iTRAQ技术筛选和鉴定造影剂急性肾损伤早期诊断标志物

发布时间：2018-04-16 22:09

本文选题：iTRAQ技术 + 造影剂急性肾损伤　；参考：《宁波大学》2017年硕士论文

【摘要】：目的:应用同位素标记相对和绝对定量(Isobaric Tags for Relative and Absolute Quantitation,iTRAQ)技术联合液相色谱-串联质谱(Liquid Chromatography tandem Mass Spectrometry,LC-MS/MS)技术筛选和鉴定经皮冠状动脉介入术(percutaneous coronary intervention,PCI)后发生造影剂急性肾损伤(contrast-induced acute kidney injury,CI-AKI)患者尿液差异蛋白,寻找CI-AKI早期诊断生物标记物。方法:收集2015年7月1日至2015年12月31在宁波市第二医院行PCI术前及术后6h、24h、48h患者尿液,根据2012KDIGO急性肾损伤(acute kidney injury,AKI)诊断标准,将患者分为:(1)CI-AKI组(PCI术后发生AKI);(2)阴性组。应用iTRAQ技术比较CIAKI组术后6h、24h、48h与术前以及CI-AKI组术后24h与阴性组术后24h的差异蛋白。将筛选出的差异蛋白按照差异倍数排序,选出每组差异倍数前10位中与CI-AKI相关的差异蛋白进行GO(Gene ontology)富集分析,了解新型生物标志物在CI-AKI可能的发生机制,并进行多组数据进行交集分析,寻找CI-AKI潜在的生物标志物。结果:48例行PCI术患者中有6例发生CI-AKI,4例男性,2例女性。(1)CI-AKI组术后6h:较术前差异蛋白151个,上调74个,下调77个;差异倍数前10位与CI-AKI相关的差异蛋白为免疫球蛋白G2重链(IGHG2)、人甘露糖结合凝集素相关丝氨酸蛋白酶2(MASP2)、血清载体蛋白A1(APOA1)、人过氧化物还原酶5(PRDX5)。(2)CI-AKI组术后24h:1)较术前差异蛋白167个,上调106个,下调61个;差异倍数前10位与CI-AKI相关的差异蛋白为癌细胞分裂周期蛋白(CDC42)、人类β防御素(HBD)、人过氧化物酶2(PRDX2)。2)较阴性组术后24h差异蛋白191个,上调77个,下调114个;差异倍数前10位与CI-AKI相关的差异蛋白为:PRDX2、水通道蛋白1(AQP1)、血清结合珠蛋白(HP)、人乳铁蛋白(LTF)、HBD。(3)CI-AKI组术后48h组:较术前差异蛋白160个,上调105个,下调55个;差异倍数前10位与CI-AKI相关的差异蛋白为AQP1、人肽酶抑制因子(PI16)。(4)通过交集分析,结果用韦恩图表示。结果显示CI-AKI组各时段与术前比较的差异蛋白以及CI-AKI组术后24h与阴性组术后24h共有的差异蛋白有14个,其中与CI-AKI相关的差异蛋白为AQP1、HBD-1、MASP2。以上都是定性(5)差异蛋白GO富集分析结果:1)分子功能:主要属于结合各种类型蛋白(包括受体)和酶的调控等功能;2)生物学过程:主要参与代谢、细胞活动和参与应激反应等过程;3)细胞定位:主要属于细胞外区域、囊泡、细胞器膜等区域。结论:(1)iTRAQ技术能够进行绝对和相对定量,检测结果精确并具有良好的可重复性,弥补了传统技术的不足。能有效筛选出CI-AKI差异蛋白。(2)AQP1、HBD-1、MASP2在CI-AKI患者尿液中含量发生显著上调,三者与CI-AKI发病机制有关,是潜在的新型生物标志物。
[Abstract]:Objective: to screen and identify acute renal contrast agent after percutaneous coronary intervention with Isobaric Tags for Relative and Absolute quantity iTRAQ and liquid Chromatography tandem Mass spectrometryLC-MS / MS technique.Urine differential proteins in patients with contrast-induced acute kidney injury-CI-AKI,To search for biomarkers for early diagnosis of CI-AKI.Methods: urine samples were collected from the second Hospital of Ningbo City from July 1, 2015 to December 31, 2015. According to the diagnostic criteria of acute kidney injury-AKI before and after PCI, the patients were divided into two groups.ITRAQ technique was used to compare the differential proteins between the CIAKI group and the pre-operation group and the CI-AKI group and the negative group at 6 hours and 24 hours after the operation.The differential proteins in the first 10 positions of each group of differential multiples were selected for GO(Gene ontology enrichment to understand the possible mechanism of the new biomarkers in CI-AKI.A number of groups of data were analyzed to search for potential biomarkers of CI-AKI.Results six of the 48 patients underwent PCI had CI-AKI in 4 males and 2 females in the CI-AKI group: compared with 151 patients with preoperative differential protein, 74 patients were up-regulated and 77 patients were down-regulated.The first 10 differential proteins associated with CI-AKI were immunoglobulin G2 heavy chain IGHG2, human mannose binding agglutinin associated serine protease 2MASP21, serum carrier protein A1 APOA1, human peroxidase reductase 5(PRDX5).(2)CI-AKI group 24 h after operation.The first 10 differential proteins related to CI-AKI were cancer cell cycle protein CDC42, human 尾 -defensin HBDG and human peroxidase 2PRDX2 路2) compared with negative group at 24 hours after operation, the differential proteins were up regulated by 77 and down regulated by 114.The first 10 differential proteins related to CI-AKI were: 1 PRDX2, 1 aquaporin 1 aquaporin, HP1, HP5, HBD. 3CI-AKI: 160 proteins, 105 up-regulated and 55 down-regulated compared with those before operation.The first 10 differentially expressed proteins associated with CI-AKI were AQP1 and human peptidase inhibitor PI16. 4) the results were expressed by Wayne map.The results showed that there were 14 differentially expressed proteins in CI-AKI group and 24 hours after operation in CI-AKI group and negative group respectively. The difference protein associated with CI-AKI was AQP1HBD-1mASP2.These are qualitative differential protein (5) differential protein enrichment analysis results: 1) molecular function: mainly belong to various types of protein (including receptor) and enzyme regulation and other functions of the biological process: mainly involved in metabolism,Cellular activity and involvement in stress response were mainly located in extracellular region, vesicle, organelle and other regions.Conclusion the technique can be used in absolute and relative quantitative analysis, and the detection results are accurate and reproducible, which make up for the deficiency of traditional technique.The CI-AKI differential protein, AQP1, HBD-1MASP2 was significantly up-regulated in the urine of CI-AKI patients, which was related to the pathogenesis of CI-AKI and was a potential new biomarker.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4;R692

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