【作者】 吴家清；

【导师】 赵明；

【作者基本信息】 南方医科大学 ， 泌尿外科， 2009， 博士

【摘要】 近二十年来,随着新型免疫抑制剂的广泛使用和配型技术的不断改善,肾移植术后急性排斥反应已得到有效控制,但移植肾的长期存活率仍无明显提高,在全世界器官来源日益短缺的严峻形势下,如何使受者来之不易的移植物长期存活,成为移植工作者当前面临的一项重要课题和紧迫任务。慢性排斥(chronic rejection,CR)是远期移植肾失功的主要原因。细胞免疫在移植肾各类排斥中的作用已经广为认可,2003年Terasaki重提器官移植体液免疫理论后,体液免疫在移植肾各类排斥中的作用也日益受到重视。目前,细胞免疫和体液免疫共同参与移植排斥的理论已经成为共识,Baff2005移植病理会议已经正式将抗体介导排斥反应的病理分类补充入Banff诊断标准中:即形态学上的组织损伤证据、补体4—d片段(completement 4d,C4d)在管周毛细血管(peritubular capillaries,PTC)的沉积、血清学检查供体特异性抗体(donor specificantibody,DSA)存在,结合临床表现,成为目前被广泛接受的移植肾排斥的诊断标准。不论是细胞免疫还是体液免疫,它们都参与了移植肾慢性排斥反应是毫无疑问的。因此术后随访过程中通过某个免疫学指标对肾移植受者准确评价其免疫状态,对预测和早期诊断慢性排斥,评估抗排斥治疗的效果及指导免疫抑制剂应用都有重要意义。移植后免疫状态的检测方法较多,这些方法从不同的侧面检测受者体内的某些免疫指标,以期反映机体整个免疫系统的功能,截至目前,仍然缺乏全面理想的监测指标。采用临床肾功能生化指标、彩超等参数对移植肾功能的监测都是非特异性的,T淋巴细胞表型的检测(CD4/CD8)虽较好地反映了机体T淋巴细胞的免疫状态,但是对体液免疫因素却不能真实反映,群体反应性抗体(panel reactive antibody,PRA)为代表的抗HLA抗体的检测反映了抗体介导的体液免疫因素,应用广泛,但对大量PRA阴性患者,仍存在无法判断其免疫状态及移植排斥风险的局限,移植肾组织穿刺活检病理学检查加上免疫组化检测C4d对移植肾慢性排斥诊断具“金标准”价值,但是程序性穿刺活检对移植肾有创伤,病人多不愿接受。因此,寻找无创的诊断排斥的免疫学指标亦是目前移植免疫研究的重要内容。Mosmann等于1986年根据产生的细胞因子及其生物学功能的不同,将鼠的辅助性T细胞(helper T cell,Th)分为Th1细胞和Th2细胞,Th1细胞通过分泌干扰素—γ(interferon-γ,IFN-γ)、白细胞介素2(Interleukin 2,IL-2)和肿瘤坏死因子-β(tumor necrosis factorβ,TNF-β)促进细胞免疫,Th2细胞通过分泌IL-4、IL-5、IL-6、IL-10、IL-13促进体液免疫。1991年Maggi等证实人的Th细胞同样划分为Th1细胞和Th2细胞,不同的是人Th1细胞和Th2细胞都可以产生IL-2;激活后的Th1细胞和Th2细胞分别表达不同的膜表面抗原,Th1细胞高表达淋巴细胞激活基因-3(LAG-3)及其编码蛋白,Th2细胞高表达CD30抗原,因此CD30抗原表达水平可反映Th2细胞的激活状态及其对免疫功能促进作用。CD30是神经生长因子/肿瘤坏死因子受体超家族中的一员,是分子量为120KD的跨膜糖蛋白,其最先在何杰金病的Hodgkin和Reed Sternberg细胞表面上发现。CD30主要表达在分泌Th2型细胞因子的CD4~+T和CD8~+T细胞上,而在分泌Th1型细胞因子的T细胞上没有或仅低表达,其胞外部分能被蛋白酶切断而释放一个分子量为88,000的可溶形式CD30(soluble CD30,sCD30)。CD30通过与存在于B细胞膜上的CD30配体结合,再与CD30~+T细胞受体(Tcell receptor)发生交联,在IL-2存在的情况下发挥作用,一方面促进Th2细胞的分化和增殖,通过IL-2等调节细胞免疫,另一方面通过分泌IL-4、IL-5等促进体液免疫。CD30~+T淋巴细胞激活的免疫机制包含了体液免疫和细胞免疫,它的具体作用原理还需要大量的研究去阐明。CD30~+T淋巴细胞被激活后,sCD30会被释放入循环体液中,可被定量或定性检测到。现有的研究已经证实,sCD30高水平表达与移植术后急性排斥发生和远期移植肾丢失风险增大有关。但是sCD30与远期移植肾慢性排斥反应的关系尚未有明确的报道。本课题通过对慢性排斥患者的血清sCD30的检测,分析其在慢性排斥中异常表达的临床意义,并同时观察PRA、C4d体液免疫指标,比较它们在慢性排斥时表达的差异。目的通过研究血清sCD30在慢性排斥患者和移植肾功能正常受者中的表达,分析移植肾慢性排斥时临床检测血清sCD30的意义,比较其与PRA和C4d检测结果的差异。方法第一部分。病例来源于珠江医院,接受尸体肾移植一年以上,以Banff2005标准确定慢性排斥组(CR组)患者36例,采用单纯随机抽样方法抽取在我院行尸体肾移植,术后一年移植肾功能正常,门诊随访的106例移植肾功能稳定受者设为对照组,慢性排斥组每一病例留取血标本和活检标本时间相同即活检时间,对照组血标本则为门诊随机随访时间留取。首先用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测两组血清sCD30,回顾性分析CR组和对照组两组间sCD30表达的差异。第二部分。移植肾穿刺活检36例CR组患者,免疫组化检测C4d,结合同时间点PRA、C4d指标,回顾性分析sCD30、PRA、C4d在慢性排斥中的表达差异。所有数据均采用SPSS13.0统计软件分析。计量资料采用(均数±标准差),采用两独立样本t检验比较CR组和正常组的均数,两组计数资料的比较采用卡方检验。通过ROC曲线分析sCD30对于慢性排斥的诊断意义,术后PRA阳性与慢性排斥的关系进行危险度分析。P≤0.05为差异有统计学意义。结果1、血清sCD30的表达与慢性排斥两组病人一般临床特征除性别有统计学差异(X~2=7.847,P=0.005),年龄、移植术前PRA、热缺血时间、冷缺血时间及术后免疫抑制剂方案方面,两组间比较均无统计学差异(P＞0.05)。慢性排斥时,CR组和对照组血清sCD30和血肌酐水平分别是,CR组血清sCD30为(193.14±18.63)U/ml,血肌酐为(220.03±28.67)umol/L;对照组血清sCD30(65.45±19.12)U/ml,血肌酐为(104.41±30.35)umol/L。两组间血清sCD30和血肌酐比较有显著性差异(t=34.982,20.019;P=0.000,0.000)。ROC曲线分析显示灵敏度特异度趋向于100%和曲线下面积趋向为1时,血清sCD30值为135.50U/ml可为后续研究的参考临界值。2、慢性排斥中sCD30表达与PRA、C4d指标关系在CR组36例病人中,按术后PRA阴性、阳性为分类标准,血清sCD30在PRA阴性病人中为(191.13±19.11)U/ml(n=15),PRA阳性病人中为(194.57±18.62)U/ml(n=21),两者比较无显著性差异(t=0.540,P=0.593);同样CR组36例病人按C4d阴性、阳性分类,血清sCD30在C4d阴性病人中为(190.24±16.27)U/ml(n=17),C4d阳性病人中为(195.74±20.61)U/ml(n=19),两者比较也无显著性差异(t=0.515,P=0.608。在对照组106例受者中,同样按术后PRA阴性、阳性分类,术后PRA阳性受者血清sCD30为(67.32±20.63)U/ml(n=22),术后PRA阴性受者血清sCD30为(64.96±18.67)U/ml(n=84),两者进行统计学比较,同样无显著性差异(t=0.515,P=0.608)。进一步分析36例CR组发现,PRA阳性21例(58.3%),C4d沉积阳性19例(52.8%),C4d阳性患者的术后PRA阳性率与C4d阴性患者的术后PRA阳性率相比,有显著性差异(84.2%vs29.4%,X~2=11.085,P=0.001)。两组受者术后PRA与慢性排斥的危险度分析得知,术后PRA阳性与术后PRA阴性相比,发生慢性排斥的优势比为5.35,95%可信区间为(2.37,12.04),诊断慢性排斥的灵敏度和特异度分别为58.3%和79.2%,总的诊断准确率为73.9%(105/142)。结论:(1)血清sCD30异常升高与慢性排斥的发生密切相关,血清sCD30检测有助于对慢性排斥发生的诊断,本研究为sCD30作为一种无创的判断慢性排斥的免疫学指标提供了初步的临床参考数据。(2)慢性排斥约半数有体液性免疫因素参加,PRA可较好地反映受体的体液免疫情况,但也存在PRA阴性时判断困难的情况,血清sCD30用于监测机体免疫状态较PRA全面,不受体液性因素影响,而与移植肾活检病理检查及C4d免疫组化染色相比,方法简单无创。更多还原

【Abstract】 In recent 20 years,with the widely using of new kinds of immune suppressants and improving of HLA matching,acute rejection after renal transplantation have been well controlled,while long-term renal graft survival still have not been increased significantly.Under the grim situation of worldwide organs shorting,it is an importment study and an urgent task of transplatation workers to improve graft survival longer.Chronic rejection is an important cause of late allograft dysfunction.Cell immunity has been recognized in all kinds of allograft rejections,in 2003, Pro.Terasaki emphasized the theory of humoral immunity again,the effect of humoral immunity on all kinds of allograft rejections has been thought highly increasingly.Nowadays,it is a common understanding that cell immunity and humoral immunity all play important roles in allograft rejections.Baff meetings of allograft pathology diagnostics has been replenished the antibody mediation rejection into the standards.No matter cell immunity or hummoral immunity,it is no doubt that they all take part in chronic rejection.Therefore,it is vital for us to evaluate the recipients immunity state and diagnose the chronic rejection at early stage.Although there are many kinds methods to detect the recipients immunity state,these indexes all have their limitations to the whole recipients immunity system.For exemple,the detect of T lymph cell CD4/CD8 may reflect the recipients T lymph cell immunity state, but it can’t tell us the information about humoral immunity.Another exemple is panel reactive antibody(PRA),it has been used to reflect the humoral immunity for many years and its clinical value has been admitted by us all,but for many many PRA negative recipients,it can not tell us the risk when the renal transplantations will be carried out.The allograft biopsy and pathological check including the C4d depsit in peritubular capillaries(PTC) is the gold standard for all kinds rejetion diagnostic,C4d is a degradation product of complement factor C4 during the classic complement pathway activated by antigen-antibody reaction and its doposit in renal graft tissure of peritubular capillaries is an established maker of humoral response in recipent,and the Banff schema has already admitted it as one of standards,but biopsy is an invasive method for recipients and allografts,so the recipients always don’t accept this diagnostic method even if the allograft function has been damaged by unknown reasons.So,it is a very immportant and impending task for us to find some noninvasive and comprehansive indexes to lean about the recipients’ immunity state.Mosmann et al classified the mouse T-helper(Th) lymph cells into Th-1 type and Th-2 type according to the cytokines and its funtion in 1986,Th1 lymph cells can secret IFN-γ、IL-2和TNF-βand promote cell immunity,Th2 lymph cells can secret IL-4、IL-5、IL-6、IL-10、IL-13 and promote humoral immunity.In 1991, Maggi et al demonstrated that human Th lymph cells also classified into Th-1 type and Th2-type according to the cytokines and its funtion,and two kinds Th lymph cells could secrete IL-2.Th2 lymph cells has the membrane antigen CD30,and Thl lymph cells has no CD30.CD30 positive Th2 lymph cells can promote two kinds immunity reaction by secrete IL-2 and so on,so the CD30 level can comprehansive reflect the recipients immunity state.The sCD30 molecule,a member of the tumor necrosis factor/nerve growth factor receptor superfamily was originally identifed as a cell surface antigen on Hodgkin’s and Reed Sternberg cells and is preferentially expressed on human CD4+ and CD8+T cells that secrete Th2-type cytokines.No or low CD30 expression was found on Th1-type cytokine-secreting T cells.A soluble form of CD30(sCD30) is released into the bloodstream after activation of CD30+T cells.In diseases such as multiple sclerosis in which Th1-type immune responsese predominate,elevated sCD30 serum levels correlated with disease remission,In diseases in which Th2-type immune responses predominate,such as lupus erythematosus or atopic dermatitis, elevated serum sCD30 was associated with increased disease activity.Furthermore, increased serum sCD30 in the early stages of HIV infection predicted a rapid progress in acquired immunodeficiency syndrome.In recent years,sCD30 has been studied that it is closely related to acute rejection and the outcomes of allografts,but there has no reports about the relationship between sCD30 and chronic rejection.Based on CD30~+T cells immunity mechanism,which includes cell and humoral immunity,and there are many unknown specific principles that need us to study,our present study investigated whether increased serum levels of sCD30 could diagnose the chronic rejection in kidney allograft,and by contrasting the indexes of PRA、C4d,we wanted to demonstrate that sCD30 was a comprehansive useful and noninvasive index for chronic rejection diagnosis.Objective:To study the relationship between sCD30 and chronic rejection,and by contrasting the indexes of sCD30,PRA and C4d,we wanted to denmonstrate that sCD30 was a comprehansive useful and noninvasive index for chronic rejection diagnosis.Methods:The first part:all recipients were from the Zhujiang Hospital of the Southern Medical University,and the renal transplant operation had been passed over one year, according to Banff pathology diagnostic standards,36 recipients were chronic rejection group(CR),and 106 recipients who had normal allografts function were selected into the control group.The blood samples of chronic group were collected while the allografts were biopsied,and the blood samples of the control group were taken randomly when the recipients came to check at our outpatient. Firstly,we detected sCD30 by the means of ELISA,then retrospectively analyzed the difference of sCD30 levels between the CR group and the control group.The statistical method was“t”test between two separate samples,The difference was significant when P≤0.05.The second part:36 cases in the CR group were all undergone allografts biopsy, and the allograft’s tissue were detected C4d by immunohistochemisty method,and at the same time the PRA were tested again,we retrospectively analyzed the differences of sCD30 levels between the PRA or C4d positive group and negative group.The statistical method were“t”test and“x~2”test between two separate groups.The difference was significant when P≤0.05.Results:1.The relationship between serum sCD30 level and chronic rejection.There were no significant differences in clinical features except gender between the CR group and the control groupThere was significant difference in sCD30 levels between the CR group and the control group,the CR group sCD30 was(193.14±18.63) U/ml,the control group sCD30 was(65.45±19.12) U/ml,the statistic values were(t=34.982,P =0.000)。According to ROC curve analyse,serum sCD30 critical piont of statistical value is 135.50U/ml.2.The relationship between serum sCD30,PRA and C4d.Among the 36 cases in CR group,15 cases were PRA negative and 21 cases were PRA positive,there was no significant difference of sCD30 levels between PRA negative and PRA positive groups.Respectively,the sCD30 were PRA(-) (191.13±19.11) U/ml and PRA(+)(194.57±18.62) U/ml(t=0.540 P=0.593＞0.05).Similarly,17 cases were C4d negative and19 cases were C4d positive among the 36 cases in CR group,there was no significant difference of sCD30 levels between the two groups.Respectively,the sCD30 were C4d(-)(190.24±16.27)U/ml and C4d(+)(195.74±20.61) U/ml,(t=0.515,P=0.608＞0.05).The further analyse of CR group,PRA(+) ratio was 58.3%(21/36), C4d(+) ratio was 52.8%(19/36),between the PRA positive rate of C4d(+) and that ofC4d(-),there was significant difference(84.2%vs 29.4%,x~2=11.085,P= 0.001)。According to the risk analyse between postopration PRA and chronic rejection in all 142 cases,compared the PRA positive cases to the PRA negtive cases, the optimistic ratio of chronic rejetion was 5.35,and the 95%confident interval was (2.37,12.04)。but the sensitivity and specificity of judgement on chronic rejection were only 58.3%and 79.2%,the total accurate rate is 73.9%(105/142)。Conclusions:(1)Abnormal elevated postoperation serum sCD30 is associated with chronic rejection,it is helpful for us to determine and predict the chronic rejetion that is happening.And our investigation may provide clinical data about sCD30 critical point.(2)Compared to PRA,serum sCD30 is relatively comprehansive in detecting chronic rejection and we have not observed the humoral influence on it. And compared to allograft biopsy and C4d test,serum sCD30 detection is noninvasive importantly.更多还原