【作者】 陈大进；

【导师】 陈江华；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 第一部分尿液可溶性T细胞免疫球蛋白粘蛋白分子-3(sTim-3)对移植肾急性排异无创性诊断及预测的价值目的：通过回顾性检测研究尿液可溶性T细胞免疫球蛋白粘蛋白分子-3(sTim-3)在肾移植患者发生急性排异时的变化,探讨sTim-3在移植肾急性排异中的诊断价值,同时进行前瞻性动态检测肾移植术后早期移植患者尿液中sTim-3的变化,进一步评估sTim-3在移植受者免疫状态监测中的作用。方法：选择2006.1-2009.10在本中心接受肾脏移植患者(有病理活检并且保留有完整尿液标本)中,术后1年内血肌酐较基础值升高25-30%以上且移植肾活检病理诊断为急性排异49例,移植肾功能稳定且术后1-2月程序活检病理诊断为移植肾状态(未见组织学损害,No-AR)58例,移植肾功能稳定且术后1-2月程序活检病理诊断为亚临床排异10例,病理证实的急性肾小管坏死9例以及慢性移植肾肾病29例。同时留取健康对照者晨尿尿标本40份。用ELISA的方法测定上述尿液sTim-3的水平(检测结果均用尿肌酐值校正),比较术后不同时间、不同组别之间sTim-3水平的差异,并用免疫组化的方法研究急性排异者、移植肾状态者Tim-3表达的差异。结果：49例急性排异患者尿液sTim-3的水平(4257±439.4,95%CI：3373-5142ng/mmol creatine)明显高于肾功能稳定患者(1433±107.2,95%CI：1218-1648ng/mmol creatine)和健康对照组患者(1269±99.3,95%CI：1068-1469ng/mmol creatine),构建诊断急性排异的ROC曲线,曲线下面积0.874(95%CI：0.8-0.948,P<0.001),结合特异性和敏感性最适组合,当sTim-3=1836ng/mmol creatinine,诊断急性排异的敏感性和特异性分别达到89.4%和82.5%,P<0.001。急性排异患者sTim-3的水平与急性肾小管坏死患者(2180±217,95%CI：1679-2680ng/mmol creatine)相比也有统计学差异(P=0.02)。在术后早期的动态观察中我们发现,急性排异组sTim-3术后一周内平均水平都在6000ng/mmol creatinine左右,而肾功能稳定组患者的sTim-3水平在术后逐渐下降,到了术后第五天左右就达到稳定状态约2000 ng/mmol creatinine左右。同时我们也发现急性排异患者肾组织中Tim-3表达增多,正常肾移植患者基本没有发现Tim-3蛋白的表达。结论：作为无创性检查的手段,首次发现尿液sTim-3在诊断急性排异,鉴别是否为耐激素排异和亚临床排异方面有较好的敏感度和特异度,该蛋白分子可能是监测移植肾免疫状态的新方法。第二部分尿液Fractalkine对移植肾急性排异无创性诊断及预测的价值目的：通过回顾性检测研究尿液Fractalkine在肾移植患者发生急性排异时的变化,探讨Fractalkine在移植肾急性排异中的诊断价值,同时进行前瞻性动态检测肾移植术后早期移植患者尿液中Fractalkine的变化,进一步评估Fractalkine在移植受者免疫状态监测中的作用。方法：选择2006.1-2009.10在本中心接受肾脏移植患者(有病理活检并且保留有完整尿液标本)中,术后1年内血肌酐较基础值升高25-30%以上且移植肾活检病理诊断为急性排异49例,移植肾功能稳定且术后1-2月程序活检病理诊断为移植肾状态(未见组织学损害,No-AR)58例,移植肾功能稳定且术后1-2月程序活检病理诊断为亚临床排异10例,病理证实的急性肾小管坏死9例以及慢性移植肾肾病29例。同时留取健康对照者晨尿尿标本40份。用ELISA的方法测定上述尿液Fractalkine的水平(检测结果均用尿肌酐值校正),比较术后不同时间、不同组别之间Fractalkine水平的差异,并用免疫组化的方法研究急性排异者、移植肾状态者Fractalkine表达的差异。结果：49例急性排异患者尿液Fractalkine的水平(429.1±56.1,95%CI:316.2-541.9ng/mmol creatine)明显高于肾功能稳定患者(94.6±8.4,95%CI:77.9-111.3ng/mmol creatine)和健康对照组患者(84.5±8.9,95%CI：66.5-102.6ng/mmol creatine),构建诊断急性排异的ROC曲线,曲线下面积0.909(95%CI：0.856-0.962,P<0.001),结合特异性和敏感性最适组合,当Fractalkine= 144.6 ng/mmol creatinine,诊断急性排异的敏感性和特异性分别达到84%和81%,P<0.001。急性排异患者Fractalkine的水平与急性肾小管坏死患者(133±9.8,95%CI：110.3-155.7ng/mmol creatine)(P<0.0001)。在术后早期的动态观察中我们发现,急性排异组Fractalkine术后三周内平均水平都维持在较高水平约400-600ng/mmol creatinine左右,明显高于肾功能稳定组患者的Fractalkine水平(100-150ng/mmol creatinine左右)。同时我们也发现急性排异患者肾组织中Fractalkine表达增多,可见多个小管的胞膜、管腔膜以及管周血管内皮细胞染成棕黄色,正常肾移植患者偶见Fractalkine表达。结论：作为无创性检查的手段,发现尿液Fractalkine在诊断急性排异中有较好的敏感度和特异度,并且能较早预测移植肾急性排异的发生。同时发生急性排异时,Fractalkine在肾小管上皮细胞和管周血管内皮细胞上的表达明显增强。第三部分尿液B细胞趋化因子-1(BCA-1)对移植肾急性排异诊断及排异类型鉴别的价值目的：通过回顾性检测研究尿液BCA-1在肾移植患者发生急性排异时的变化,探讨BCA-1在移植肾急性排异及排异类型中的诊断价值,同时进行前瞻性动态检测肾移植术后早期移植患者尿液中BCA-1的变化,进一步评估BCA-1在移植受者免疫状态监测中的作用。方法：选择2006.1-2009.10在本中心接受肾脏移植患者(有病理穿刺并且保留有完整尿液标本)中,术后1年内血肌酐较基础值升高25-30%以上且移植肾活检病理诊断为急性排异49例,移植肾功能稳定且术后1-2月程序活检病理诊断为移植肾状态(未见组织学损害,No-AR)58例,以及慢性移植肾肾病29例。同时留取健康对照者晨尿尿标本40份。用ELISA的方法测定上述尿液BCA-1的水平(尿标本的数据均用尿肌酐值校正),比较术后不同时间、不同组别之间BCA-1水平的差异。结果：49例急性排异患者尿液BCA-1的水平(8.1±2.1,95%CI：3.9-12.4pg/μmol creatine)明显高于肾功能稳定患者(0.65±0.3,95%CI：0.044-1.26pg/μmol creatine)和慢性移植肾肾病组患者(2.2±0.8,95%CI；0.69-3.78pg/μmol creatine),健康对照组患者几乎都低于检测低限。构建诊断急性排异的ROC曲线,曲线下面积0.881(95%CI：0.811-0.951),结合特异性和敏感性最适组合,当BCA-1=0.23pg/μmol creatinine,诊断急性排异的敏感性和特异性分别达到81.6%和87.9%。我们还发现急性体液性排异患者的BCA-1的水平(24.2±6.7pg/μmol creatinine)显著高于急性细胞性排异患者的水平(2.9±0.6pg/μmol creatinine),P=0.009。构建诊断急性体液性排异的ROC曲线,曲线下面积0.867(95%CI：0.725-1.005),结合特异性和敏感性最适组合,当BCA-1=8.3pg/μmol creatinine,诊断急性体液性排异的敏感性和特异性分别达到75%和91.9%。在术后早期的动态观察中我们发现,急性排异组BCA-1术后3周内平均水平都维持在2 pg/μmol creatinine以上,而肾功能稳定组患者的BCA-1水平都维持在2 pg/μmol creatinine以下。结论：应用无创性检测尿液BCA-1水平的方法,首次为临床提供了一个非常有用的辅助诊断指标,通过观察BCA-1水平的变化,我们能较好地诊断移植肾急性排异以及急性排异的类型。更多还原

【Abstract】 Kidney transplantation has become the best replacement therapy for patients with chronic renal failure, one-year survival rate after transplantation has improved markedly. Graft failure from immunologic factors and death from opportunistic infection in the first year after transplantation are no longer common clinical outcomes other than 30 years ago. However, the long-term outcome of the graft is still poor because of allograft dysfunction after transplantation. Intensity of acute rejection and effect of treatment have direct impact on the long-term outcome of the graft. It is crucial that a definite diagnosis should be as early as possible. How to detect rejection and distinguish it from other causes of renal dysfunctionn is still an challenging topic in the transplantation field.With the application of new immunosuppressive agents, more and more patients presented with atypical clinical manifestation of acute rejection, Histopathologic examination of renal biopsy is regarded as the gold standard of acute rejection. But biopsy is limited by the invasive nature of the procedure and cost. Morever, because of the inadequate sample size and sampling error, there have been conflicting results of the histological findings. Some patients with stable renal function were detected with abnormal pathological presentation in protocol biopsies. Current Banff criteria is not sufficient for clinical practice in some atypical cases. To target individualized immunosuppressive regemin, it is important to evaluate the immune state of this special population.In recent years, researchers have been dedicated to explore more sensitive and timely indicators for the diagnosis of early renal allograft dysfunction. Many noninvasive test supplementing the biopsy has been used for monitoring acute rejection. For example, noninvasive tests in kidney transplants by measure of Mig, IP 10, Tim-3, Fractalkine in urine.The aim of this study is to investigate whether level of sTim-3, Fractalkine and BCA-1 in urine is a useful noninvasive tool for the assessment of acute rejection in renal transplant recipients. Our topic is expected to provide new noninvasive dignostic marks for acute renal rejection and to provide some clinical basis for future research.Part I Noninvasive Diagnostic and Predictive Value in Renal Transplant Recipients by Measurement of urine sTim-3Objective:To investigate the relationship between early-stage renal acute rejection and the level of sTim-3 in urine, explore the diagnostic value and noninvasive monitoring in early stage after transplantation by measurement of urine sTim-3.MethodsWe examined urine samples from renal transplant patients between January 2006 and October 2009.155 patients were enrolled, including 49 with biopsy-proved acute rejection,58 patients with stable renal function and no abnormal histological findings, 10 patients with subclinical rejection in protocol biopsy,9 patients with biopsy-proven acute tubular necrosis and 29 patients with biopsy-proven chronic allograft nephropathy. Additionally, urinary samples were also collected from 40 healthy controls. sTim-3 was measured in urinary samples using a commercial human sTim-3 enzyme-linked immunosorbent assay (ELISA) kit (GBD Systems). All samples were tested in duplicate by a standardized ELISA technique. Statistical analysis was performed by using SPSS software package. (Version 16.0) Because of nonparametric distribution, comparisons of sTim-3 levels among different groups were performed by Kruskal-Wallis test or Mann-Whitney U test and Wilcoxon Signed Ranks Test. A conventional receiver operating characteristic (ROC) curve was applied to determine the sensitivities and specificities for sTim-3 measurements for patients with acute rejection, subclinical rejection and steroid-resistant acute rejection. Area under the curve was also calculated. Results were expressed in the text as mean±SEM unless otherwise stated. P<0.05 was considered statistically significant. sTim-3 levels were expressed per millimole of urinary creatinine to correct for difference in urinary concentration. Immunohistochemistry for Tim-3 expression was performed on biopsies from renal transplant patients with acute rejection and No-AR.ResultsPatient with acute rejection excreted urinary sTim-3 at a significantly higher level (4257±439.4,95%CI:3373-5142 ng/mmol creatine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis excreted urinary sTim-3 at a significantly lower level (2180±217,95%CI:1679-2680 ng/mmol creatine) than levels of patients with acute rejection. ROC curve was constructed to determine the discriminatory power of sTim-3 levels for diagnosis of acute rejection. The area under ROC curve was 0.874 (95%CI:0.8-0.948), which showed that sTim-3 was a suitable marker for the diagnosis of acute rejection. At a cut point of 1836 ng/mmol creatinine, the sensitivity was 89.4% and the specificity was 82.5%(P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary sTim-3 concentration than patients with steroid-sensitive acute rejection (5548±613.5,95%CI: 4287-6809 ng/mmol creatinine vs 2653±391.7,95%CI:1830-3476 ng/mmol creatinine, P=0.0002).The dynamic level of urinary sTim-3 in acute renal rejection patients within 3 weeks after transplantation is about 6000 ng/mmol creatine, which is remarkably higher than patients with stable renal function (2000 ng/mmol creatinine). We also find that sporadic expression of Tim-3 was present on biopsies from patients with acute rejection and none in patients with No-AR.ConclusionsThe monitoring of sTim-3 in urine may be a new and noninvasive approach for detection acute rejection as well as useful to predict response to antirejection therapy. It has good sensitivity and specificity. Besides, measurement of sTim-3 in urine is a simple, inexpensive method for the routine clinical monitoring after kidney transplantation.PartⅡNoninvasive Diagnostic and Predictive Value in Renal Transplant Recipients by Measurement of urine FractalkineObjectiveTo investigate the relationship between early-stage renal acute rejection and the level of Fractalkine in urine, explore the diagnostic value and noninvasive monitoring in early stage after transplantation by measurement of urine Fractalkine.MethodsWe examined urine samples from renal transplant patients between January 2006 and October 2009.155 patients were enrolled, including 49 with biopsy-proved acute rejection,58 patients with stable renal function and no abnormal histological findings, 10 patients with subclinical rejection in protocol biopsy,9 patients with biopsy-proven acute tubular necrosis and 29 patients with biopsy-proven chronic allograft nephropathy. Additionally, urinary samples were also collected from 40 healthy controls. Fractalkine was measured in urinary samples using a commercial human Fractalkine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). All samples were tested in duplicate by a standardized ELISA technique. Statistical analysis was performed by using SPSS software package. (Version 16.0) Because of nonparametric distribution, comparisons of Fractalkine levels among different groups were performed by Kruskal-Wallis test or Mann-Whitney U test and Wilcoxon Signed Ranks Test. A conventional receiver operating characteristic (ROC) curve was applied to determine the sensitivities and specificities for Fractalkine measurements for patients with acute rejection, subclinical rejection and steroid-resistant acute rejection. Area under the curve was also calculated. Results were expressed in the text as mean±SEM unless otherwise stated. P<0.05 was considered statistically significant. Fractalkine levels were expressed per millimole of urinary creatinine to correct for difference in urinary concentration. Immunohistochemistry for Fractalkine expression was performed on biopsies from renal transplant patients with acute rejection and No-AR.ResultsPatient with acute rejection excreted urinary Fractalkine at a significantly higher level (429.1±56.1,95%CI:316.2-541.9 ng/mmol creatine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis excreted urinary Fractalkine at a significantly lower level (133±9.8,95%CI:110.3-155.7 ng/mmol creatine) than levels of patients with acute rejection. ROC curve was constructed to determine the discriminatory power of Fractalkine levels for diagnosis of acute rejection. The area under ROC curve was 0.909 (95%CI:0.856-0.962), which showed that Fractalkine was a suitable marker for the diagnosis of acute rejection. At a cut point of 144.6 ng/mmol creatinine, the sensitivity was84% and the specificity was 81%(P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary Fractalkine concentration than patients with steroid-sensitive acute rejection (526.8±85.5,95%CI:351-702.5 ng/mmol creatinine vs 309.6±81.6,95%CI: 135.7-483.4 ng/mmol creatinine, P=0.01).The dynamic level of urinary Fractalkine in acute renal rejection patients within 3 weeks after transplantation fluctuated during 400-600 ng/mmol creatine, which is remarkably higher than patients with stable renal function (100-150 ng/mmol creatinine). We also find that widespread expression of Fractalkine was present on biopsies from almost patients with acute rejection and few patients with No-AR.ConclusionsAs a noninvasive monitoring, Fractalkine in urine may be a new approach for detection acute rejection as well as useful to predict response to antirejection therapy. It has good sensitivity and specificity. Besides, measurement of Fractalkine in urine is a simple, inexpensive method for the routine clinical monitoring after kidney transplantation.Part III Noninvasive Diagnostic and Predictive Value in Renal Transplant Recipients by Measurement of urine BCA-1ObjectiveTo investigate the relationship between early-stage renal acute rejection and the level of BCA-1 in urine, explore the diagnostic value and noninvasive monitoring in early stage after transplantation by measurement of urine BCA-1.MethodsWe examined urine samples from renal transplant patients between January 2006 and October 2009.155 patients were enrolled, including 49 with biopsy-proved acute rejection,58 patients with stable renal function and no abnormal histological findings,9 patients with biopsy-proven acute tubular necrosis and 29 patients with biopsy-proven chronic allograft nephropathy. Additionally, urinary samples were also collected from 40 healthy controls. BCA-1 was measured in urinary samples using a commercial human BCA-1 enzyme-linked immunosorbent assay (ELISA) kit ((R&D Systems). All samples were tested in duplicate by a standardized ELISA technique. Statistical analysis was performed by using SPSS software package. (Version 16.0) Because of nonparametric distribution, comparisons of BCA-1 levels among different groups were performed by Kruskal-Wallis test or Mann-Whitney U test and Wilcoxon Signed Ranks Test. A conventional receiver operating characteristic (ROC) curve was applied to determine the sensitivities and specificities for BCA-1 measurements for patients with acute rejection, acute humoral rejection and steroid-resistant acute rejection. Area under the curve was also calculated. Results were expressed in the text as mean±SEM unless otherwise stated. P<0.05 was considered statistically significant. BCA-1 levels were expressed per micromole of urinary creatinine to correct for difference in urinary concentration.ResultsPatient with acute rejection excreted urinary BCA-1 at a significantly higher level (8.1±2.1,95%CI:3.9-12.4 pg/μmol creatine) than levels of patients with No-AR and healthy controls (P<0.001). Patients with acute tubular necrosis excreted urinary BCA-1 at a significantly lower level (2.52±0.57,95%CI:1.21-3.84pg/μmol creatinine) than levels of patients with acute rejection. ROC curve was constructed to determine the discriminatory power of BCA-1 levels for diagnosis of acute rejection. The area under ROC curve was 0.881 (95%CI:0.811-0.951), which showed that BCA-1 was a suitable marker for the diagnosis of acute rejection. At a cut point of 0.23 pg/μmol creatine, the sensitivity was 81.6% and the specificity was 87.9%(P<0.001). Patients with steroid-resistant acute rejection had significantly greater urinary BCA-1 concentration than patients with steroid-sensitive acute rejection (11±2.82,95%CI:5.22-16.8 pg/μmol creatinine vs 5.34±4.28,95%CI:3.8-14.5 pg/μmol creatinine, P=0.0032). Patients with acute humoral rejection had significantly higher urinary BCA-1 concentration than patients with acute celluar rejection (24.2±6.68,95%CI:9.5-38.9 pg/μmol creatinine vs 2.91±0.65,95%CI:1.6-4.22 pg/μmol creatinine, P=0.0002). ROC curve was constructed to determine the discriminatory power of BCA-1 levels for diagnosis of acute humoral rejection. The area under ROC curve was 0.867 (95%CI:0.725-1.005), which showed that BCA-1 was a suitable marker for the diagnosis of acute humoral rejection. At a cut point of 8.3 pg/μmol creatine, the sensitivity was 75% and the specificity was 91.9% The dynamic level of urinary BCA-1 in acute renal rejection patients within 3 weeks after transplantation is above 2 pg/μmol creatine, however, the level of patients with stable renal function is below 2 pg/μmol creatine.ConclusionsThe monitoring of BCA-1 in urine may be a new and noninvasive approach for detection acute rejection as well as useful to discriminate the type of rejection. It has good sensitivity and specificity.更多还原