摘要
急性排斥反应(acute rejection,AR)是影响移植肾长期存活的一个关键因素。也是引起早期移植物损害和长期的移植物功能障碍以及慢性移植物肾病的主要原因,而目前通常诊断肾移植术后发生急性排斥反应方法包括:1.病人临床体征,2.血肌酐、尿素氮等非特异性生化指标检查,3.移植肾影像学检查,4.移植肾穿刺病理活检。前3项检查指标只有当移植肾功能发生明显损伤后才能出现改变,并且这些改变都是非特异性的。组织活检虽然是确诊急性排斥反应的“金标准”,但穿刺活检可能导致严重的并发症,而由于活检取材部位的限制,并不能及时、全面的反应出移植肾的损伤情况。因此,目前临床急需筛选具有前瞻性、非侵入性和特异性的早期诊断AR的方法,以指导临床用药,提高移植肾的长期存活率
尿液可以及时准确地反映整个肾脏的生理状态和功能水平,而且收集简单方便、是非侵入性诊断肾脏疾病的理想标本。器官移植排斥反应的发生是一种复杂的、动态的、多因素的病理过程,很难用传统单一的指标来准确反映。而蛋白质组是动态的,有它的时空性,能够在生命有机体整体水平上阐明生命现像的本质和活动规律。因此本课题以肾移植术后发生AR前、中、后各期的尿液作为研究对象,应用二维凝胶电泳-质谱技术(2-DE-MS)及Westernblot技术等,以期找到一组具有前瞻性和特异性的蛋白分子作为早期诊断的候选标志物。
首先,我们根据尿蛋白的含盐高的特点优化了2-DE过程中等电聚焦程序,与Bio-rad操作手册推荐的等电聚焦方法相比,获得的尿液2-DE图谱点数由476个增加到564个。为充分去除尿液中的盐分,我们建立了尿蛋白丙酮沉淀三步法处理样品,结果显示尿蛋白丙酮沉淀三步法处理得到的样本在上述优化的等电聚焦条件下所获得的分离蛋白点更加清晰且圆,水平条带更少。说明本次研究建立的2-DE技术平台适用于尿蛋白研究。
然后,为了消除尿蛋白样品的个体差异,本研究采取的是移植排斥反应前后患者自身样品的比较,即:排斥前3天相对光密度值与排斥后21天相对光密度值相比,选择表达量变化大于3倍(P﹤0.05),并根据两个体取6个时相点即:-3/-2/-1/7/14/21天相对光密度值做趋势分析,应同时具有明显下降趋势的30个蛋白点进行鉴定。经MADIL-TOF-MS/MS成功鉴定出其中的16个蛋白,经文献复习、生物信息学和蛋白功能检索,从中筛选了与免疫排斥反应相关的3个蛋白,分别为α-1抗胰凝乳蛋白酶(alpha-1-antichymotrypsin ,AACT),锌-α2糖蛋白(Zn-Alpha-2-Glycoprotein,ZAP)和肿瘤排斥抗原gp96(tumor rejection antigen gp96,gp96),进行进一步临床样本免疫印记验证。
对肾移植术后发生AR患者临床确诊排斥前3～5天(n=8)和未发生AR患者术后7天(n=10)尿液蛋白进行Westrenblot验证发现:以肉眼观察印记条带出现有无为判断标准,三个蛋白判定的准确率、敏感性和特异性分别为:AACT, 77.78%/100%/60%;ZAG, 77.78%/100%/60%;gp96, 83.33%/87.5%/80%。将三个蛋白各组的测定值与同组对照的比值,运用LINGO8.0软件进行统计学处理,得到三个蛋白的判断理论临界值[0.15, 0.35, 0.50],以此界值作为判别标准时,单一的ZAG可以将准确率、敏感性和特异性提高到100%/100%/100%,AACT准确率、敏感性和特异性分别达到94.4%/100%/90.00%;gp96准确率、敏感性和特异性分别达到88.9%/100%/90.00%。最后应用统计学建立优化模型对三个蛋白联合进行判别时,与原始病例实际结果的符合率达100%,但其确切的诊断价值还有待多中心大样本的临床试验确认。
综上所述,我们的研究结果提示,联合应用AACT、ZAG和gp96三个蛋白临界值可作为早期诊断肾移植急性排斥反应的指标。
Acute rejection, a key point to the long-term survive of renal grafts, is the major reason for the early damage and long-term dysfunction of the grafts and chronic allograft nephropathy. General diagnosis of acute rejection after renal transplantation includes four aspects as follows: (1)clinical manifestation;(2)laboratory test, some nonspecific biochemical indicator such as serum creatinine and urea nitrogen; (3)imageolgy examination, for example, color Doppler ultrasonography; (4)pathological biopsy. The first three tests, which are not characteristic for diagnosis, are obviously hysteretic so that the precise diagnosis would be made after the allografts had already suffered from acute rejection and been severely damaged. Biopsy, although thought to be the golden rule for the acute rejection final diagnosis, is an invasive way which probably causes terrible complications. Owing to the limited location for acquiring the sample, the whole status of the allografts can not be detected. Thus, early diagnosis biomarkers of acute rejection, which are prospective, noninvasive and specific are emergency requested to guide the clinical medication for the sake of increasing the survival rate of grafts.
Urine reflects the physiological function and can be conveniently and safely collected, which is a perfect sample for non-invasive diagnosis of renal disease. Organ transplantation rejection is a complicated, dynamic and multiple factor procedure, which can not be accurately diagnosed by traditional unitary index. Proteomics which is dynamic and various in different time and status can systemically reflect the essence and rule of biological phenomenon. Thus we took the urine of pre-, mid- and post-AR as sample; find a series of candidate protein which might be meaningful for the diagnosis of acute rejection via two-dimensional gel electrophoresis mass spectrometry and initially validated these proteins.
Firstly, we optimized the ampholine electrophoresis procedure since the urine protein had high electrolytes by which the protein dots number in 2-DE map was increased from 476 to 564 and the boosting pressure procedure was simple in contrast to the bio-rad recommended protocol. Meanwhile, in order to minimize the influence of electrolytes on 2-DE, we constructed a new way to acquire urine protein, namely acetone tripartite precipitation, by which the protein dots number in 2-DE pattern was average 761 which was obviously more than average 521 by acetone one step precipitation and the shape of the dots were distinct and circular, not to mention the level strap was apparently reduced.
Secondly, in order to eliminate the individual difference between the urine protein samples, we choose two victims who suffered AR postoperative and compared the 2-DE map on different time points, which was three days, two days and one day before the clinical diagnosed AR (-3d,-2d,-1d)and the 7th,14th ,21st day after clinical diagnosed AR. Then we applied image analysis software to quantitate the 2-DE map and from the comparison of the 2-DE pattern on three days and two days before and the 21st day after the clinical diagnosed AR, we picked up the protein which was three times changed (P﹤0.05)and had the similar diversity trend in the two victims and identified them via mass spectrum.
A total of 16 differential protein spots were successfully identified by MALDI-TOF-MS. Analyzed by means of bioinformatics and protein function index, alpha-1-antichymotrypsin (AACT), Zn-Alpha-2-Glycoprotein (ZAP) and tumor rejection antigen gp96 (gp96) were considered as candidate proteins for the further verification via immunoblotting. Four groups were established: urine from both of AR patients (n=8, 3-5days before clinical definition of AR) and stable patients (n=10, 7days after transplantation) after renal transplantations, urine from normal people (n=2) and operative patients of other diseases (n=2, 3days after operation) as control. Judgment from the imprinting straps by macroscopic observation, the accuracy rate, sensibility and specificity of these three proteins were: AACT, 77.78%/100%/60%; ZAG, 77.78%/100%/60%; gp96, 83.33%/100/80%. In view of these unsatisfied standers, we argued that protein existed or not is improper diagnosis stander, and the notable change of protein expression is also significant for diagnosing the disease. Therefore, the quantitative change of the aim proteins were analyzed by LINGO8.0 software, the accuracy rate, sensibility and specificity of the proteins were increased to 94.4%/ 100%/90.00%; 100%/100% 100%/ and 88.9%/100%/90.00%, respectively. Due to the limitation by sample volume, the study of multi-center clinical trials should be further carried out.
In summary, our data suggest that early diagnosis of kidney graft acute rejection based on supposed criteria of AACT, ZAG and gp96 could be the promising in future.
引文
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