摘要
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 Non-invasive detection is the focus of intense research in diagnosis of acute renal allograft rejection currently. Urine protein is considered the cue to reflect the pathological changes in kidney disease. In this study, we explored the urine markers for early acute renal allograft rejection. Methods The urine protein of two patients with acute renal allograft rejection were examined by 2D gel electrophoresis and bioinformatics. We adopted pH 4 -7 ready strip IPG and stained the gel with Sypro-Ruhy. The digitized 2D maps of urine protein were quantitatively analyzed using 2D-analysis software packages. By analyzing the differential expressions of proteome between different time points （ 1, 2, 3 days before acute rejection and 7, 14, 21 days after acute rejection）, 30 protein spots were selected and analyzed by MALDI-TOF-MS/MS. Results We obtained 2D gel electrophoresis maps of urine protein of the patients with acute renal allograft rejection, which are of good reproducibility and resolution. Sixteen protein spots were identified, resulting in thirteen corresponding proteins. Out of these proteins, we screened three proteins （ alpha-l-antichymotrypsin, tumor rejection antigen gp96, Zn-Alpha-2-Glycoprotein） closely related to acute rejection. Conclusion The urine protein spots on 2D gel electrophoresis maps for the patients with acute renal allograft rejection were of obvious difference when detected at different time points of acute rejection. Alpha-l-antichymotrypsin, tumor rejection antigen gp96 and ZnAlpha-2-Glycoprotein might be the candidate protein markers to diagnose acute renal allograft rejection after renal transplantation.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第24期2267-2271,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(30500447)~~
关键词
肾移植
急性排斥反应
二维电泳
质谱
kidney transplantation
acute rejection
two dimensional gel electrophoresis
mass spectrum
