【作者】 杨振林；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2005， 博士

【摘要】 临床器官移植的长期存活率得到明显提高,已成为终末期器官功能衰竭病人的最佳治疗方案。然而,同种异体排斥反应仍然是器官移植的主要障碍。诱导机体抗原特异性免疫耐受,是解决器官移植排斥的根本方法。 目前,尚无系统的免疫学和免疫遗传学的对移植耐受预测和判断的方法,临床上采用组织活检鉴别移植物的免疫状态虽然准确,但相对机体的免疫状态的变化有明显的滞后性,并且标本的获得严重受限。在器官移植术后常规运用大剂量的免疫抑制剂治疗方案,带来了严重的副作用,如肾毒性、肝毒性、骨髓抑制等各器官系统的直接损害;病人免疫力降低会导致的各种感染和恶性肿瘤发病率上升等严重的后果。所以,如何建立预测器官移植受体免疫耐受的方法,为临床移植病人用药个体化提供依据,对耐受病人用药减量或停药及时给以指导,改善病人预后和生活质量,减轻经济负担,对免疫排斥的早期诊断及病人的预后作出判断,这对自发耐受发生率较高的肝脏移植的意义尤为重要。 1986年,mosmann等发现CD4+T辅助细胞可产生Th1型(IL-2、IFN-γ)利Th2型(IL-4、IL-10)细胞因子。最近有研究表明,Th1和Th2型细胞因子表达的变化和免疫耐受的产生和维持以及排斥反应的发生具有非常密切的关系。 此课题应用短期小剂量CsA诱导大鼠同种异体肝移植达到长期接受和耐受的移植模型,拟在研究Th细胞在外周血的表达和移植肝脏中的表达是否存在相似性,能否作为肝移植术后急性排斥和耐受的预测指标。浙江人学博」:学位论文材料和方法 实验为同种异体移植:I组为BN斗BN大鼠同基因对照组(Syn group);工工组为Lewis oBNCSA处理组(CsA group),大鼠肝移植后用环抱霉素A(CsA)2.omg/kg/d皮下注射0一7天;IH组为Lewis分BN人鼠急性排斥组(Rej group)。移植后1、3、5、7、14天切取移植肝脏用于病理学、移植肝脏中基因产物的逆转录聚合酶链式反应和原位杂交反应检测;受体脾用于淋巴细胞亚群激光共聚焦显微镜检测;受体外周血用于淋巴细胞亚群的流式细胞检测、血清细胞因子EL工SA的检测。另外设亚组观察移植后一般情况和肝移植术后受体的存活时间。 结果1.各组大鼠术后一般状态及生存时间 同基因组,均长期存活,(大于100天);CsA处理组大鼠,6只中4只获得长期存活,用药期间一般状态差,停药后好转;排斥组大鼠术后28一38天全部死亡,中位存活时间33.3天。同基因组和CsA处理组术后一般状态明显好于排斥组。2.病理表现及移植排斥反应病理分级 同基因组移植大鼠各个时间段病理学检测均未发现明显排斥。CsA处理组第l大未见有排斥表现,第3天可见淋巴细胞浸润,平均排斥分级分别为0.48,第5、7天淋巴细胞细胞浸润更加明显,平均排斥分级分别为0.67和l.ro,14大淋巴细胞浸润,胆管增生存在,平均排斥分级为1.16。排斥组术后第l大汇管区少量炎性细胞浸润,术后第3天汇管区淋巴细胞细胞浸润明显,可见肝实质变性,平均排斥分级为1.59级,术后第5天中度排斥表现,平均排斥分级为2.34级,术后第7天和14天,出现严重急性排斥表现,静脉内皮炎明显,大量炎性细胞浸润,可见肝的变性坏死,胆管内皮炎或小胆管消失,平均排斥分级分别为2.87不[一2.93。 除第1天外,CsA处理组各时间段的排斥分级明显高于同基因组(p<0.05或<0 .01):而排斥组排斥分级和同基因组及CsA处理组相比升高更加明显(p<0 .05或<0 .01)。CsA处理组不11排斥组内排斥分级比较p<0.001。3.流式细胞仪检测外周血.CD4‘CD45RC细胞结果浙江人学博l:学位论文3.1 CD4+CD45RC.细胞在外周血单核细胞百分比中的变化 csA处理组从第3天开始升高,第14大达到高峰,高于同基因组,和同基因组相比有明显差别(p<0 .001),第100天回到了手术第1大的水平。排斥组从第3天逐步升高,高于同基因组和CsA处理组,与之相比差别有显著性(p刁.047、0.034),第5、7、14大分别和同基因组和CsA处理组相比差异更明显(p<0.001)。3.2 CD4‘CD45RC细胞在外周血单核细胞一百分比中的变化 CD4‘CD45RC细胞在外周血单核细胞中的一百分比在同基因组只有术后第3天有轻度的升高,第5、7、14、100大接近术后第1大的水平。排斥组从第3天升高,然后逐步轻度下降。CsA处理组从第5大突然升高,高于其他组,和同基因组和排斥组组相比有明显的差别(p=0 .018、0.025);第7天达到高峰,和同基因组和排斥组组相比有明显差别(p=、<0 .001);第14天较第7天有所下降,但是和同基因组和排斥组组相比仍较高,有明显的差别(p<0 .001);第100大进一步下降,仍高于同基因组,但无显著性差别。4.激光共聚焦显微镜检测CD4‘CD45RC细胞在脾淋巴细胞中的表达 每个高倍视野脾细胞中CD4CD45RC细胞数量变化:在同基因组的术后第1、3、5、7、14的表达在4一6士1个;在CsA处理组第1、3、5、7、14大的表达分别为4士1、6士1、7土1、9士2、9士l个;在排斥组第l、3、5、7、14大的表达分别为6士l、9士2、12士2、16士3、13士2个。5.Thl细胞因子在基因水平和外周血.中的表达5.1工FN一丫在移植肝组织内mRNA水平和外周血.中的表达 IFN一Y在移植肝组织内mRNA水平和外周血.中的表达有极大相似性,同基因组在各时段均未有表达。CsA处理组在1、3、5更多还原

【Abstract】 IntroductionSignificant enhancement in long-term livability of clinic organ transplantation makes it an optimal therapy protocol for treatment of end stage disease. However, allogeneic rejection is still a major problem in organ transplantation. Inducing immunotolerance may be the essential method to overcome rejection reaction in organ transplantation.At present, there is no systemic predictability and estimation method for transplantation tolerance from the point of immunology and immunogenetics. Although clinically used biopsy provide accuracy results, but it display a significant hysteresis for immune state changes of organism. Meanwhile, there is a limitation in obtaining specimen. Conventional adopted large dosage immunosuppression therapy procedure post organ transplantation lead to serious side effects, such as direct damage to organ systems of kidney toxicity, liver toxicity and marrow suppression et al., various infection and morbidity increase of malignancy et al. resulted from decreased immunity of patients. Consequently, developing expectation method for accepter immune tolerance of organ transplantation to provide basis for clinically personal administration of transplantation patients, reducing or stopping administration for tolerance patients, improving patient prognosis and live quality, decreasing financial burden, early diagnosis to immunorejection and guiding prognosisjudgment of patient, this have significant influences on liver transplantations that have higher probability for spontaneous tolerance occurrence.In 1986, Mosmann et al. reported that CD4+ T cell can produce Th1 type (IL-2、 IFN-γ) and Th2 type (IL-4, IL-10) cytokines. Recent studies suggest that changes expressed by Thl and Th2 type cytokines are closely related with the appearance and maintaining of immunotolerance and occurrence of rejection reaction.Inducing allogeneic rejection transplantation liver of rat, to obtain long-term accepted and tolerance transplantation model, was achieved by administration of minimal dose CsA in our laboratory. The aim is to study whether there is similarity between expression of Th cell in peripheral blood and transplantation liver, whether it can be used as an estimation parameter for acute rejection and tolerance post liver transplantation.Materials and MethodsOrthotopic rat liver transplantation was performed. (Brown-Norway) BN rats were used as syngeneic donors and Lewis (LEW) were used as allogeneic donors. All recipients were BN rats who were randomly divided as follows: Group I: Syngeneic control (BN-to- BN); Group II: Acute rejection treated with CsA 2mg/kg/d by subcutaneous injection from day 0-7. Group III: Acute rejection (LEW -to- BN).A11 groups were subdivided into day 1, 3, 5, 7,14(n=4 each) posttransplantation respectively for sample harvesting; and additional subgroups (n=6) for observation of general situation and survival time. Liver graft specimens were harvested at indicated time to determine morphological changes by pathological examination and gene expression by reverse transcription-polymerase chain reaction (RT-PCR), as well as the hybridization in situ. Recipient splenocytes were isolated and phenotypes of spleen T cell subsets were analyzed by confocal microscopy. Recipient T cell subsets in peripheral blood were analyzed by flow cytometry and blood serum cytokines were analyzed by Enzyme-linked immunoadsordent assay (ELISA) .Results1. The general situation and survival time posttransplantationThe general situation of syngeneic group was very well and survival time was over 100 days after transplantation. As for CsA treated group, the general situation was bad during CsA medication but with only mild rejection and the four of six recipients survived over 100 days .in the acute rejection group, all recipients died from 28 to38 days after transplantation and the mean survival time was 33.3 days. The general situation of syngeneic group and CsA treated group was remarkably better than that of acute rejection group.2. Histopathologic examination and rejection gradeNo signs更多还原