【作者】 徐莹；

【导师】 陈江华；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 前言肾移植已经成为终末期肾病的最佳替代治疗方法,但术后肾功能不全的诊断和治疗将影响到移植物及受体的存活率。与30年前相比,肾移植术后1年内由于免疫性因素发生移植物失功以及机会性感染导致受者死亡已经不再是普遍临床现象,但早期诊断排异并且区分于其它原因导致的。肾功能障碍仍然没有取得突破性的研究成果。同时,随着新型免疫抑制剂的应用,部分临床诊断排异且抗排异治疗有效的患者的移植肾穿刺活检病理表现并不典型,还有部分患者临床肾功能正常,但在常规程序性活检时病理有异常表现。为了达到个体化的免疫抑制剂方案,对这些受体的免疫状态进行准确客观的评估将是一个新的课题。近年来,各种通过基因、细胞、蛋白等不同水平的无创性监测手段的应用使术后早期肾功能不全的诊断更敏感、及时,虽然目前无创性的血、尿检查筛选到的检测诊断指标不少,但没有一个指标能得到完全公认并被常规应用于临床实践。目前临床上仍然把血肌酐水平的升高作为移植物功能障碍的常规检测标记,但导致血肌酐水平升高的原因有很多,为了进一步鉴别诊断,有创性的移植肾病理活检和无创性的影像学检查是临床常规开展的另两项检查手段。尽管存在一定的局限性,目前移植物功能评价的金标准仍然是病理活检,而且对急性排异的诊断主要依据移植物内细胞的浸润情况,尤其是T淋巴细胞在诊断中的地位不可忽视。本课题通过免疫组化的方法检测移植物内调节性T细胞功能标记物FOXP3及细胞毒T细胞功能标记物Granzyme B的表达情况,结合临床资料,分析其在移植物免疫状态评估中的作用,希望通过分子病理学的角度对现有的病理诊断标准起到一定的辅助作用。此外,本课题还首次在中国人群中前瞻性地研究了血氧水平依赖的功能磁共振成像(BOLD-MRI)在移植肾术后早期急性排异中的诊断和预测作用。该检查技术通过监测移植肾内不同的氧合情况判断术后早期肾功能障碍的原因,具有无创性、可重复性、连贯性以及敏感性较好的优点,提供了一个从病理生理学的角度诊断移植肾急性排异的无创性手段。本课题希望在现有临床常规开展的功能学和组织学方法诊断急性排异的基础上,从分子免疫学和病理生理学的角度进一步探讨相关技术对急性排异的诊断价值,为以后的研究提供一定的临床基础。第一部分移植肾活检标本内浸润T细胞表型分析对急性排异的诊断价值目的对临床诊断排异,且抗排异治疗有效,但移植肾活检病理表现未达到急性排异诊断标准的移植物内浸润T细胞免疫表型进行分析,与活检证实的急性排异以及程序性活检病理表现正常组进行比较,判断其免疫状态趋向于排异还是耐受,希望对现有的病理诊断标准起到一定的辅助作用。方法共入选活检穿刺标本175份,穿刺时间均在移植术后1年内,其中活检证实的T细胞介导的急性排异组67例;临床诊断排异组63例,包括病理诊断为临界改变组51例,移植肾状态伴炎细胞浸润组12例;肾功能正常术后1—2月内程序性活检病理诊断为移植肾状态伴炎细胞浸润,随访至今肾功能一直正常组45例。免疫组化方法检测同一活检标本连续切片内调节性T细胞表面标记物FOXP3、细胞毒性T细胞表面标记物Granzyme B的表达情况,定量计数阳性细胞数目,同时记录每张切片内FOXP3阳性T细胞/Granzyme B阳性T细胞比值。结果正常组100%(45/45)均表现为以调节性T细胞表面标记物FOXP3为主的耐受表型:活检证实的急性排异组94.03%(63/67)表现为以细胞毒性T细胞表面标记物Granzyme B为主的排异表型,临床诊断排异病理诊断为临界改变组86.27%(44/51)表现为以细胞毒性T细胞表面标记物Granzyme B为主的排异表型(与活检证实的急性排异组相比,P=0.204),临床诊断排异病理诊断为移植肾状态伴炎细胞浸润组83.33%(10/12)表现为以细胞毒性T细胞表面标记物Granzyme B为主的排异表型(与活检证实的急性排异组相比,P=0.224)。结论活检证实的急性排异组和临床诊断排异组均表现为以细胞毒性T细胞为主的排异表型,移植物内FOXP3阳性T细胞/Granzyme B阳性T细胞比值可作为诊断急性排异的辅助分子病理学指标。第二部分移植肾程序性活检标本内浸润T细胞表型分析对移植物免疫状态的监测作用目的对临床肾功能正常,术后1—2月内接受程序性活检的标本内浸润T细胞免疫表型进行分析,探讨相关免疫分子表达情况对移植物功能及预后的影响。方法共入选100例临床肾功能正常,术后1—2月内接受移植肾程序性活检的标本,包括亚临床排异组10例,临界改变组29例,移植肾状态伴炎细胞浸润组61例。免疫组化方法检测同一活检标本连续切片内调节性T细胞表面标记物FOXP3、细胞毒性T细胞表面标记物Granzyme B的表达情况,定量计数阳性细胞数目,同时记录每张切片内FOXP3阳性T细胞/Granzyme B阳性T细胞比值。统计术后1年内的临床随访情况,评估程序性活检时移植物内免疫表型对移植物术后1年内临床情况的预测价值。结果与临床肾功能异常组相比,大部分程序性活检标本中浸润的T细胞表现为以FOXP3标记的调节性T细胞为主的免疫耐受表型,但其中亚临床排异组中有2例(20%),临界改变中有5例(17.2%),移植肾状态伴炎细胞浸润组中有8例(13.1%)表现为以Granzyme B为标记的细胞毒T细胞为主的排异表型,该部分受者在穿刺后1年内均出现免疫活动现象,包括活检证实的排异以及临床诊断排异,或者是肌酐水平增高,加大免疫抑制剂剂量后恢复正常。结论肾功能稳定的程序性活检标本虽然表现出一定的细胞浸润或临界改变甚至亚临床排异,但浸润T细胞免疫表型仍以调节性T细胞为主的耐受表型;移植物内FOXP3阳性T细胞/Granzyme B阳性T细胞比值可反映活检当时移植物内免疫状态,对移植物临床预后有一定的预测作用,可作为调整免疫抑制剂方案的预警信号。第三部分血氧水平依赖的功能磁共振成像对早期移植肾排异的诊断和预测作用目的前瞻性研究血氧水平依赖的磁共振成像(BOLD-MRI)在移植肾急性排异中的诊断和预测作用,同时探讨其在急性排异和肾小管坏死中的鉴别诊断作用。方法纳入2005年12月—2007年3月在本中心首次接受同种异体尸体肾移植患者共110例,分为肾功能正常组82例,活检证实的急性排异组21例,活检证实急性肾小管坏死组7例,记录3组的基线资料,采用血氧水平依赖的磁共振成像(BOLD-MRI)技术进行影像学检查,测量间接反映移植肾组织氧生物利用情况的磁共振参数R2*。结果急性肾小管坏死组平均皮质R2*值为15.25±1.03/s,明显高于肾功能正常组(13.35±2.31/s,P=0.028)和急性排异组(12.02±1.72/s,P=0.001),急性排异组平均皮质R2*值明显低于肾功能正常组(P=0.013);急性排异组平均髓质R2*值为14.02±2.68/s,明显低于正常组肾功能正常组(16.66±2.82/s,P＜0.001)和急性肾小管坏死组(19.47±1.62/s,P＜0.001),急性肾小管坏死组平均髓质R2*值明显高于肾功能正常组(P=0.011);相关分析显示除了平均动脉压与平均皮质R2*值之间有相关性(P=0.029),其余临床资料均不影响R2*值水平;ROC曲线分析显示,皮、髓质R2*值均可作为急性排异和急性肾小管坏死的辅助诊断指标:肾功能正常组中低水平髓质R2*值(MR2*＜14.9/s,23例)者日后发生急性排异的比率高于高水平髓质R2*值(MR2*≥14.9/s,59例)者,但未达到统计学差异(17.39%vs 8.47%,P=0.259)。结论肾移植术后早期急性排异及急性肾小管坏死时肾内氧合状态发生显著变化;BOLD-MRI技术可作为肾移植术后早期急性排异的辅助诊断手段,并可能有一定的预测价值,同时对急性排异和急性肾小管坏死的鉴别诊断具有一定价值。更多还原

【Abstract】 IntroductionKidney transplantation has become the best replacement therapy for patients with chronic renal failure,however,allograft dysfunction after transplantation has negative impact on graft and patient survival.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,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,some patients with typical clinical manifestation of acute rejection presented with atypical pathological change; Meanwhile,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.Although several non-invasive monitoring indicator were confirmed to be effective,none of them was recognized and applied regularly in clinical practice as an independent indicator.A rise in the level of serum creatinine is used as the indicator of allograft dysfunction regularly in clinical practice,to aid in the resolution of the difficult differential diagnosis of allograft dysfunction,invasive renal biopsy and noninvasive imaging technqiue are often performed regularly in clinic.Until now,pathological diagnosis is still the golden criteria to evaluate the function of renal allograft irrespective of its limitations.Infiltration of mononuclear cells, especially T lymphocytes has predomindant status in the Banff criteria.In this study, we analyze the immune phenotype of T lymphocyte infiltration in allograft biopsies, explore the immue state evaluation value of FOXP3(functional marker of regulatory T cells)and Granzyme B(functional marker of cytotoxic T cells)in combination with clinical data.We hope the molecular pathological study will be of supplemented value for present pathological diagnosis criteria.Meanwhile,we prospectively study the diagnosis value of blood oxygen level-dependent magnetic resonance imaging(BOLD-MR/)by assessing the oxygenation state of renal transplants in acute rejection,find it is a non-invasive diadynamic imaging technique with good sensitivity and specificity in the diagnosis of allograft dysfunction early after kidney transplantation.It has dignostic value of acute renal rejection in pathophysiological respect.Our topic is expected to provide new dignostic methods for acute renal rejection in terms of molecular immunology and pathophysiology on the basis of histological and functional method which are now used regularly in clinical practice,and to provide some clinical basis for future research. PartⅠImmune phenotye analysis of T lymphocytes infiltration in renal biopsies for the diagnosis of acute renal rejectionObjectiveTo analyze the immune phenotype of T lymphocytes infiltration in renal biopsies with typical clinical manifestation of acute rejection but atypical pathological change (clinical rejection),compared with normal group and biopsy-proved acute rejection,to judge the immune state of this special population.Methods175 specimens were enrolled,including 67 with biopsy-proved acute T cell mediated rejection,63 with clinical rejection,consisted of 51 with borderline change and 12 with only inflammatory cell infiltrations but no obvious pathological abnormalities, and 45 with normal pathological presentations but inflammatory cell infiltrations in protocal biopsies with stable renal function in our follow-up time.Detect the expression of FOXP3(functional marker of regulatory T cells)and Granzyme B (functional marker of cytotoxic T cells)by the means of immunohistochemical methods in serial slices of the same biopsy.Quantitation of positive cell number and the ratio of FOXP3-positive T cells/Granzyme B-positive T cells were recorded simultaneously.Results100%(45/45)of normal group presented with regulatory phenotype dominated by FOXP3-positive T lymphocytes,94.03%(63/67)of biopsy-proved acute T cell mediated rejection group presented with cytotoxic phenotype dominated by Granzyme B-positive T lymphocytes;Borderline change group presented with similar phenotype compared with BPAR group(86.27%vs 94.03%,P=0.204);only inflammatory cell infiltrations but no obvious pathological abnormalities group also presented with similar phenotype compared with BPAR group(83.33%vs 94.03%,P=0.224).ConclusionsRenal biopsies with typical clinical manifestation of acute rejection but atypical pathological change(clinical rejection)have similar immune phenotype of T cell infiltration compared with biopsy-proved T cell mediated acute rejection.The ratio of FOXP3(functional marker of regulatory T cells)-positive T cells to Granzyme B (functional marker of cytotoxic T cells)-positive T cells could be a valuble diagnostic molecular indicator in pathological criteria. PartⅡImmune phenotye analysis of T lymphocytes infiltration in protocol renal biopsies for the evaluation of immune stateObjectiveTo analyze the immune phenotype of T lymphocytes infiltration in protocol renal biopsies with stable renal function within 2 months after transplantation,and to explore the evaluation value of related immune markers.MethodsAll specimens were obtained in protocal biopsies,100 specimens were enrolled, including 10 with subclinical rejection(SCR group),29 with borderline change (Pro-BL group)and 61 with only inflammatory cell infiltrations but no obvious pathological abnormalities(Pro-N group).Detect the expression of FOXP3(functional marker of regulatory T cells)and Granzyme B(functional marker of cytotoxic T cells) by the means of immunohistochemical methods in serial slices of the same biopsy. Quantitation of positive cell number and the ratio of FOXP3-positive T cells/Granzyme B-positive T cells were recorded simultaneously.Clinical data during the follow-up time were also recorded.ResultsCompared with indicated biopsies,the majority cases presented with regulatory phenotype dominated by FOXP3-positive T lymphocytes in protocal biopsies; however,20%(2/10)in SCR group,17.2%(5/29)in Pro-BL group and 13.1%(8/61) in Pro-N group presented with cytotoxic phenotype dominated by Granzyme B-positive T lymphocytes;All these 15 allografts developed different type of abnormalities in functon within 1 year after biopsy,including biopsy-proved acute rejection,clinical diagnostic acute rejection,and fluctuation of serum creatinine level which was improved by adding amount of immunosuppressive agents.ConclusionsAlthough with abnormal pathological changes according to Banff criteria,the majority cases in protocol biopsies presented with regulatory phenotype dominated by FOXP3-positive T lymphocytes;The ratio of FOXP3(functional marker of regulatory T cells)-positive T cells to Granzyme B(functional marker of cytotoxic T cells) -positive T cells could evaluate the immune state of allograft in protocol biopsy and predict the clinical outcome,it could be an early-warning indicator to guide the adjustment of immunosuppressive regimens. PartⅢDiagnosis and prediction of early acute renal transplant rejection with blood oxygen level-dependent MR imagingObjectiveTo prospectively assess the oxygenation state of renal transplants using blood oxygen level-dependent magnetic resonance imaging(BOLD-MRI)to diagnose and predict early acute renal rejection,and differentiate between acute rejection and acute tubular necrosis.MethodsBOLD MR imaging was performed in a cohort of 110 patients undergoing cadaver renal transplants between Dec 2005 and March 2007,82 had clinically normal functioning transplants,21 had biopsy-proved acute rejection and 7 had biopsy-proved acute rejection.Cortical R2*(CR2*)and medullary R2*(MR2*)levels of transplanted kidneys were measured.ResultsThe mean CR2~* level was significantly higher in the ATN group(15.25±1.03/s) compared to the normal group(13.35±2.31/s,P=0.028)and AR group(12.02±1.72/s,P=0.001).There was a significant difference also between the AR group and normal group on CR2~* levels(P=0.013).The mean M R2~* level was significantly lower in the AR group(14.02±2.68/s)compared to the normal group(16.66±2.82/s,P＜0.001)andATN group(19.47±1.62/s,P＜0.001).There was also a significant difference between the ATN group and normal group on M R2~* levels(P= 0.011).There were no correlations between characteristics such as patient age, post-operation time,post-biopsy time,Scr level,HB level,urine output volume,MAP level,CNI trough concentration and R2~* levels,except between MAP level and CR2~* level(P=0.029);ROC curve analyses suggested that cortical and medullary R2~* values could accurately discriminate acute rejection and acute tubular necrosis from normal function in the early posttransplant period;In the normal functioning transplants group,those with lower medullary R2~* values(MR2~*＜14.9/sec,n=23)had higher acute rejection rates than those with higher medullary R2~* values (MR2~*＞14.9/sec,n=59)in the first 6 months posttransplant,but the difference between two groups was not significant(17.39%vs 8.47%,P=0.259).ConclusionsThere were siginificant tissue oxygen bioavailability change during the episodes of acute rejection and acute tubular necrosis.BOLD-MRI could be a non-invasive dynamic imaging technique with good sensitivity and specificity,and may have the forecast value;It could also be a valuable method to discriminate between acute rejection and acute tubular necrosis.更多还原