【作者】 范凯；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 胸外科， 2013， 博士

【摘要】 第一部分：建立小鼠闭塞性细支气管炎模型实验背景：近年来众多学者建立了多种动物模型来研究肺移植术后阻塞性细支气管炎(obliterative bronchiolitis,OB)的发病机制。在本研究中,我们在移植术后一段时间内比较几种常见的OB模型的特点。实验方法：受体小鼠分组后分别接受原位气管移植,大网膜下气管异位移植以及皮下气管异位移植。气管移植物分别在术后14、21以及28天,进行组织学或者免疫组织化学评估。实验结果：移植术后,来自不同移植部位的同种气管移植物基本保持了正常的组织学结构,而与各移植部位相应的异种移植物的气道出现明显的阻塞,同时在各个实验观测时间点时伴有更多的CD4+/CD8+单个核细胞核肌纤维细胞的浸润,但是新生的上皮细胞核新生血管明显减少(P<0.05)。与两种异位气管异种移植物相比较,原位异种移植物的气管的管腔相对通畅,伴有明显较少的CD4+/CD8+单个核细胞核肌纤维细胞的浸润,但是却有明显更多的新生上皮细胞和新生血管形成(P<0.05)。实验结论：根据我们的实验结果提示,小鼠原位气管移植可以被用作研究0B发展早期病变的模型,而异位气管移植可以作为OB晚期的研究模型。第二部分：上皮向间质转化在小鼠气管移植模型中的作用研究背景：在患有细支气管闭塞综合征(Bronchiolitis Obliterans Syndrome, BOS)的肺移植患者的植肺中,常出现气道正常上皮损伤,而大量纤维疤痕组织增生的气道重构。在本研究中,我们假设小鼠气管原位移植模型中移植气管上皮在出现阻塞性气道疾病时出现上皮向间质转化(epithelial-mesenchymal transition, EMT)。实验方法：受体小鼠分为同种异体(Balb/C to C57BL/6)以及同种自体(Balb/C to Balb/C)组,分别接受原位气管移植。移植术后受体小鼠均不使用免疫抑制剂治疗。将受体气管移植物分别在术后14、21以及28天进行组织学或者免疫组织化学评估。实验结果：移植术后,同种自体气管移植物组织学结构基本保持正常,与之相比同种异体气管移植物的管腔较为狭窄(P<0.05)。术后与同种自体气管移植比较,同种异体气管移植物气道上皮内正常上皮标志物E-cadherin表达较低,而间质标志物α-SMA增高,上皮损伤以及EMT标志物MMP-9表达增高(P<0.05)。实验结论：我们实验结果表明,在小鼠气管移植模型中OAD的发生伴有气道上皮向间质转化的现象。因此提供治疗OB的可能靶点。第三部分：雷帕霉素抑制小鼠气管移植物中EMT的发生,减少阻塞性气道疾病实验背景：雷帕霉素(RAPA)作为免疫抑制剂用来治疗多种实体器官免疫排斥反应。雷帕霉素能改善肺移植患者术后肺功能,但是由于诸多不良反应,许多患有BOS的肺移植患者无法耐受雷帕霉素的治疗。在本实验中,我们试图研究小剂量雷帕霉素对同种异体小鼠气管移植物的治疗效果。实验方法：受体小鼠分组后分别接受原位气管移植,术后根据分组给予不同剂量的雷帕霉素+助溶剂(实验组)或者单独给予助溶剂(载体对照组)。各组受体小鼠的气管移植物分别在移植术后第28天,进行组织病理学、免疫组织化学、Western-Blot以及流式细胞学评估。实验结果：与各实验组比较,载体照组中同系异体小鼠气管移植物的气道管腔明显狭窄(P<0.05),上皮发生明显的上皮向间质转化：E-Cadherin表达降低(P<0.05),α-SMA以及MMP-9表达升高,移植气管上以及外周血中纤维细胞的表达水平均增高(P<0.05)。中、高剂量RAPA组中治疗效果无统计学差异(P>0.05),切均强于小剂量的RAPA治疗效果(P<0.05)：外周血中纤维细胞的表达水平无统计学差异,但移植气管上纤维细胞表达水平均低于小剂量治疗组。实验结论：根据我们的实验结果表明,较大剂量的雷帕霉素能够通过抑制EMT发生来减少气道OAD的发生,移植气管上皮中EMT在OAD发生过程中其重要作用。更多还原

【Abstract】 Part Ⅰ:Establishment of Murine Obliterative Bronchiolitis ModelBackground:Couples of animal models have been developed to investigate the mechanisms of obliterative bronchiolitis (OB) post lung transplantation. In this study, we compared three prevalent murine models of obliterative bronchiolitis in terms of several pathologic changes after transplantation.Methods:Recipient mice received one of the orthotopic, intra-omental and subcutaneous tracheal transplantations in both syngeneic (Balb/C to Balb/C) and allogeneic (Balb/C to C57BL/6) settings. No immunosuppressive drugs were administered. Tracheal grafts were harvested on Day14,21and28post-transplant for histological and immunohistochemical assessment.Results:Syngeneic tracheal grafts from different transplant sites maintained normal histologic structures, while the corresponding allografts exhibited more occlusion of the airway lumen as well as more infiltration of CD4+/CD8+mononuclear cells and myofibroblasts, but less regenerative epithelium and neovascularized vessels at indicated times (P<0.05). Compared with the two heterotopic allografts, the orthotopic had less occlusion of the tracheal lumen as well as less CD4+/CD8+mononuclear cells and myofibroblasts, but more regenerative epithelium and neovascularized blood vessels (P<0.05).Conclusions:We presume orthotopic tracheal transplantation in mice can act as a model to study early stages of OB, and heterotopic tracheal transplantation can be a model for late stages of OB. Part Ⅱ:Epithelial-Mensenchymal Transition in Murine Orthotopic Tracheal AllograftBackground:In graft lungs of patients with BOS, airway remodelling was observed, including epithelium of the airway shedding and fibrosis proliferation. In this study, we hypothesized that epithelial-mesenchymal transition (EMT) occurs with the development of BOS after transplantation.Methods:Recipient mice received orthotopic tracheal transplantation in both syngeneic (Balb/C to Balb/C) and allogeneic (Balb/C to C57BL/6) setting. No immunosuppressive treatment was administered. Tracheal grafts were harvested on Day14,21and28after transplantation for histological and immunohistochemical analyses.Results:Syngeneic tracheal graft maintained normal histological structures, while allografts exhibited more occlusion of the airway lumen (P<0.05). Compare with syngeneic tracheal grafts, tracheal allografts exhibited lower expression of epithelium markers E-cadherin, but higher expression of myofibroblasts markers a-SMA as well as marker of epithelial damage and EMT MMP-9(P<0.05).Conclusions:Our data demonstrates that EMT occurs with the development of Obstructive airway disease in mice tracheal allografts,which suggests that EMT is a potential target in the treatment of OB post lung transplantation. Part III:Rapamycin Inhibits Epithelial-Mesenchymal Transition and Attenuates Obliterate Airway Disease in Murine Tracheal AllograftBackground:Rapamycin (RAPA) is employed to prevent rejection of transplanted solid organ as an immunosuppressive agent. Rapamycin can improve pulmonary function of lung transplant patients with bronchiolitis obliterans syndrome (BOS), but as a result of severe adverse events, it is sometimes intolerant. In this study, we investigate the outcomes of murine tracheal allografts treated by reduced-dose Rapamycin.Methods:Recipient mice underwent orthotopic tracheal transplant after been allocated into randomized groups. Recipients were treated with either different doses of Rapamycin (experimental group) or solvent (control group) after transplantation. Tracheal allografts were harvested in the28th day post-transplant for histological, immunohistochemical, Western-Blot and Flow Cytometry analyses.Results:Compared with tracheal allografts in experimental groups, tracheal allografts in control group exhibited more occlusion of the airway lumen (P<0.05) and underwent epithelium to mesenchymal transition:lower expression of E-Cadherin, but higher expression of myofibroblasts markera-SMA, MMP-9as well as fibrocytes both in peripheral blood and tracheal allografts (P<0.05). The outcomes of mid-dose and high-dose RAPA groups made no differences (P>0.05), which were better than those in low-dose RARA groups:the cell counts of peripheral fibrocytes in all groups were not significantly different (P>0.05), but the cell counts of tissue fibrocytes in mid-dose RAPA groups were both decreased than those in low-dose group (P<0.05).Conclusions:Our study demonstrated that higer-dose Rapamycin can attenuate obstructive airway disease with inhibiting epithelium-mesenchymal transition in murine tracheal allograft, and EMT plays an import role in development of obstructive airway diseases post tracheal transplantation.更多还原