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目的利用二维凝胶电泳和生物信息学技术,从严格筛选的2例肾移植术后发生急性排斥反应患者尿液标本中,寻找急性排斥反应早期标志物。方法在pH值为4～7范围,采用Sypro-Ruby染色,对发生急性排斥反应过程的不同时间点(-3,-2,-1,7,14,21d)的患者尿液二维凝胶电泳(Two-dimensional Gel Electrophoresis,2-DE)图谱进行比较,挑选了30个有上调或者下调趋势的蛋白点。将这些蛋白点进行切胶、胰酶消化后用MALDI-TOF-MS/MS分析。结果得到了重复性好、分辨率高的肾移植术后尿蛋白2-DE图谱。30个差异蛋白点经质谱分析和数据库检索,鉴定了16个蛋白点,对应13种蛋白质。根据蛋白功能查询结果,发现了3个蛋白含量变化趋势与排斥反应密切相关的蛋白,分别是α-1抗胰凝乳蛋白酶(alpha-1-antichymotrypsin,AACT)、肿瘤排斥抗原gp96(tumor rejection antigen gp96)和锌-α2糖蛋白(Zn-Al-pha-2-Glycoprotein)。结论肾移植术后发生急性排斥反应患者的不同时间点尿液2-DE图谱存在明显差异。AACT、肿瘤排斥抗原gp96和锌-α2糖蛋白可能作为临床诊断肾移植术后急性排斥反应的标志物候选蛋白。
Objective To detect the early markers of acute rejection from 2 urine samples of patients with acute rejection after renal transplantation by using two-dimensional gel electrophoresis and bioinformatics techniques. Methods Sypro-Ruby staining was performed in the range of pH 4 ~ 7 for two-dimensional urine of patients with different time points (-3, -2, -1, 7, 14 and 21d) Electrophoresis (Two-dimensional Gel Electrophoresis, 2-DE) comparison of the map, picked up 30 spots with up or down trend. The spots were excised and analyzed by MALDI-TOF-MS / MS after trypsinization. RESULTS: Urinary protein 2-DE maps of renal allografts with good reproducibility and resolution were obtained. Thirty differential protein spots were identified by mass spectrometry and database searches, identifying 16 protein spots corresponding to 13 proteins. According to the results of protein function search, three proteins closely related to rejection were found, including alpha-1-antichymotrypsin (AACT), tumor rejection antigen gp96) and Zn-Al-pha-2-Glycoprotein. Conclusions There is a significant difference in urine 2-DE at different time points after acute rejection in patients after renal transplantation. AACT, tumor rejection antigen gp96, and zinc-α2 glycoprotein may serve as biomarker candidates for the clinical diagnosis of acute rejection following renal transplantation.