【作者】 齐战；

【导师】 王善政；

【作者基本信息】 山东大学 ， 外科学， 2005， 博士

【摘要】 实验一 同种异体大鼠嵌合体动物模型的建立及意义 目的 探讨建立同种异体大鼠嵌合体动物模型的方法,初步研究嵌合体与免疫耐受的关系。方法 对受体Wistar大鼠（♀）采用两种诱导方法:Ⅰ组大鼠亚致死TBI（11Gy）;Ⅱ组大鼠TBI（7Gy）,两组均在4h内输入SD大鼠（♂）BMC（8×10⁷）,Ⅱ组于2d后腹腔注射CTX（50mg/kg）。分别于BMT20d、40d后通过PCR方法检测SD大鼠源性BMC在Wistar大鼠体内植活情况,并通过皮肤移植、DTH和MLR检查判断其耐受情况。结果 经上述处理两组大鼠外周血均检测出SD大鼠源性嵌合体,皮肤移植、DTH和MLR检查显示对SD大鼠产生特异性耐受,但Ⅰ组大鼠生存情况明显不如Ⅱ组。结论 应用7GyTBI+腹腔注射CTX（50mg/kg）+供体BMT可成功建立同种异体大鼠嵌合体动物模型,并诱导特异性免疫耐受。 实验二 肺移植后OB动物模型及发病机制的初步探讨 目的 建立肺移植OB动物模型,并对其发病机制进行初步探讨。方法 SD大鼠气管（每段5环）异位移植到SD大鼠（Ⅰ组）和Wistar大鼠（Ⅱ、Ⅲ组）腹腔并用大网膜包裹,Ⅰ、Ⅱ组不接受免疫抑制药物,Ⅲ组全程接受CsA(10mg·kg^-1·d^-1)。分别在移植后3d、14d、28d取出移植气管检测其组织形态学改变和Th1（IL-2、IFN-γ）/Th2（IL-4、IL-10）细胞因子表达,同时观察CsA对上述指标的影响。结果 同种异体大鼠气管腹腔异位移植模型大体上复制了人类肺移植后OB的组织病理学变化。仅仅在同种异体移植物发生的闭塞性气道疾病（OAD）分为三个阶段:开始是缺血期（同系和同种异体移植物均能观察到）,随后是淋巴细胞浸润期,上皮几乎完全丢失（仅在同种异体移植物观察到）,最后是闭塞期,同种异体移植物管腔纤维性闭塞。Ⅱ组Th1（IL-2、IFN-Y）/Th2（IL-4、IL-10）细胞因子表达比Ⅰ组明显升高。与Ⅰ组相比,Ⅱ组Th1细胞因子表达比Th2细胞因子升高更为显著。与Ⅱ组比较,CsA（Ⅲ组）明显减少了管腔阻塞和淋巴细胞浸润,但对于移植物上皮没有明显保护作用。Ⅲ组移植物IL-2表达比Ⅱ组明显减少,但两组IFN-γ、IL-4、IL-10更多还原

【Abstract】 EXPERIMENT 1: ESTABLISHMENT AND EVALUATION OF ALLOGENIC CHIMERA ANIMAL MODEL IN RATS.Objective To establish the allogeneic mixed chimera model in rats and explore the mechanism of tolerance induced by chimera. Methods Wistar rats (recipient, ♀) were conditioned by two methods: Rats were induced with sublethal total body irradiation (TBI) in Group I (11Gy) and with TBI in group Ⅱ (7Gy), followed by infusion of bone marrow cells (8× 10~7) of SD (donor, ♂ )rats within 4 hours, then rats in group II were administered cytoxan (CTX, 50mg/kg) intraperitoneally 2 days later only. Recipient rats were detected for donor origin cells in the peripheral blood lymphocyte on day 20, 40 by polymerase chain reaction (PCR). To explore tolerance the mechanism, skin grafting, mixed lymphocyte reaction (MLR) and delayed type hypersensitivity (DTH) were performed. Results Chimera of SD rats can be found in the peripheral blood lymphocyte of the tolerance Wistar rats in Group I and II. Wistar rats were specifically tolerant to SD rats in skin graft, DTH and MLR assays, but wistar rats in group I have worse survival compared with that in group Ⅱ. Conclusion Treatment of 7Gy TBI and injection of CTX(50mg/kg) plus donor bone marrow transplantation (BMT) can successfully establish rat chimera model and induce a specific tolerance in Wistar to SD rat.EXPERIMENT 2: ESTABLISHMENT OF AN ANIMAL MODEL OF OBLITERATIVE BRONCHIOLITIS AFTER LUNG TRANSPLANTATION AND DETECTION OF ITS PATHOGENESIS PRELIMINARILY.Objective The purpose of this study was to establish an animal model of obliterative bronchiolitis (OB) after lung transplantation and investigate the pathogenesis of it preliminarily. Methods Tracheal segments (5 cartilaginousrings each) were transplanted from SD rats to SD rats (isografts: Group I) or to Wistar rats (allografts: Group II and III). Grafts were implanted into an abdominal cavity and wrapped in the omentum. Animals in Group I and II did not receive immumosuppressive drugs, animals in Group III received CsA daily by gastrotube (gastrogavage) at lOmg.kg’.d"1 from beginning to end. Grafts were harvested at day 3,14 ,28 after transplantation as representative time points for three phases of injury in the evolution of allograft airway obliteration, then examined histological changes and gene expression of T-helper 1[ Thl: interleukin-2 (IL-2), interferon-gamma (IFN-y) J-and T-helper 2[ Th2: interleukin-4 (IL-4), interleukin-10 (IL-10) ]-type cytokines in gafts. At the same time, effects of CsA were observed on the above-mentioned indices. Results This heterotopic tracheal transplant rat model described in this report replicated the histopathological lesion seen in obliterative bronchiolitis (OB) following clinical lung transplantation. The obliterative airway disease (OAD) named for this animal model developed only in allografts, which showed a triphasic time course: an initial ischemic phase (observed in both isografts and allografts) was followed by a marked lymphocytic infiltrative phase with complete epithelial loss (observed only in allografts), and finally by an obliterative phase with fibrous obliteration of the allograft airway lumen (observed only in allografts). In Group II (allografts), expression of IL-2, IFN-y, IL-4, and IL-10 were much higher than that in Group I (isografts). Expression of Thl cytokines was increased to a greater extent than that of Th2 cytokines in Group II compared with Group I. In Group III, CsA reduced the development of fibroproliferation and lymphocyte infiltration markedly than that in Group II, but it did not protect the epithelium. Allografts in Group III expressed significantly less IL-2 gene transcripts than that in Group II over all the points. There was no significant difference between Group II and III in IFN-y, IL-4, and IL-10 gene expression. Conclusions Althouh differences exist between the evolution in this animal model and the chronic rejection process in human lung transplant recipients, these findings reproduce the characteristic features of obliterative bronchiolitis and demonstrate that this lesion result from allograft rejection. It will be useful for studying the pathogenesis, prevention, and treatment of obliterative bronchiolitis after lung transplantation. Thl and Th2 lymphocyte subtypes contribute to the development of obliterative bronchiolitis in heterotopic trachea transplant model of rat, and changes of their cytokines gene expression may be involved inthe pathogenesis. CsA inhibits IL-2 gene transcripts, so it can reduce development of the pathologic lesion of obliterative bronchiolitis to a certain degree.EXPERIMENT 3: CHIMERA INDUCTION BY BONE MARRROW TRANSPLANTATION AND ITS EFFECTS ON OBLITERATIVE BRONCHIOLITIS AFTER LUNG TRANSPLANTA TION.Objective The purpose of this study was to induce chimera by donor bone marrrow transplantation and observe its effects on obliterative bronchiolitis (OB) after lung transplantation, and investigate the pathogenesis of transplantation tolerance of chimera preliminarily in an animal model. Methods Chimera was induced by donor bone marrrow transplantation before tracheas of SD rats were transplanted into the abdominal cavity of Wistar rats (an animal model of OB). Grafts were harvested at day 14 and 28 after transplantation, observed histological changes which involved epithelium, lumen and lymphocytic infiltration, and examined gene expression of Thl (IL-2> IFN-y) and Th2 (IL-4> IL-10) type cytokines in gafts. Results At day 14, the epithelium in Group I had almost regenerated to normal appearance, lymphocytic infiltration and luminal obliteration were lowest, too. The epithelium regenerated evidently in Group IV, which was better than that in Group II or III. At the same time, lymphocytic infiltration and luminal obliteration were lower in Group IV than that in Group II or III (p<0.01). These changes were more evident at day 28. In Group I, isografts had almost no expression of IL-2 and IFN-v> expression of IL-4 and IL-10 was lowest, too. Expression of IL-2, IFN-y, IL-4 and IL-10 was lower in Group IV compared with that in Group II or HI. There were significant differences between Group I and Group II or III (p<0.01), especially for IL-2, IFN-v. Conclusions Induction of chimera by donor bone marrow transplantation (BMT) can induce specific transplantation tolerance successfully and reduce development of the pathologic lesion of OB significantly. It may be one of the mechanism that lymphocytic infiltration and expression of Thl and Th2 cytokines decreased.更多还原