雷帕霉素减轻小鼠气管移植物上皮向间质转化以及阻塞性气道疾病的实验研究

发布时间：2018-03-21 23:07

本文选题：肺移植　切入点：阻塞性细支气管炎　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05),上皮发生明显的上皮向间质转化：E-Cadherin表达降低(P0.05),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(P0.05)。中、高剂量RAPA组中治疗效果无统计学差异(P0.05),切均强于小剂量的RAPA治疗效果(P0.05)：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。
[Abstract]:Part one: establishing a mouse model of obliterans bronchiolitis
Background: in recent years, many animal models have been established by many scholars to study the pathogenesis of obliterative bronchiolitis (OB) after lung transplantation. In this study, we compared the characteristics of several common OB models after transplantation.
Experimental methods: the recipient mice were grouped into orthotopic tracheal transplantation, subtotal tracheal heterotopic transplantation and subcutaneous tracheal heterotopic transplantation respectively. Tracheal grafts were evaluated histologically or immunohistologically at 14,21 and 28 days after operation.
Results: after transplantation, allogeneic transplantation from different parts of the tracheal grafts remain normal histological structure, and the corresponding with the transplantation site of xenografts was airway obstruction, at the same time in each experimental observation time point with more CD4 +/CD8+ infiltration of mononuclear cell muscle fiber cells. But the newborn newborn vascular epithelial cells was significantly reduced (P0.05) plant. Compared with two kinds of heterotopic tracheal xenotransplantation, orthotopic xenograft tracheal lumen is relatively smooth, with significantly fewer infiltrating mononuclear cell CD4+/CD8+ muscle cells, but significantly more new epithelial cells and neovascularization (P0.05).
Conclusion: according to our experimental results, tracheal transplantation in mice can be used as a model for early development of 0B, and ectopic tracheal transplantation can be used as a late OB research model.
The second part: the role of epithelial mesenchymal transition in the mouse model of tracheal transplantation
Background: in patients with bronchial occlusive syndrome (Bronchiolitis Obliterans, Syndrome, BOS) and lung transplantation patients, often appear normal airway epithelial injury, and airway remodeling of fibrous scar tissue hyperplasia. In this study, we assume that the gas pipe in the mice model of orthotopic transplantation Transplantation in obstructive airway epithelium airway disease occurs when the epithelial to mesenchymal transition (epithelial-mesenchymal transition, EMT).
Experimental methods: the recipient mice were divided into allograft (Balb/C to C57BL/6) and autologous (Balb/C to Balb/C) group, underwent orthotopic tracheal transplantation. Transplantation recipient mice were not using immunosuppressive therapy. The plant after the operation. The 14,21 and 28 days for histological or immunohistochemical evaluation will shift the recipient trachea.
Results: after transplantation, autologous tracheal graft histology remained normal, compared with the lumen of tracheal allograft graft is narrow (P0.05). Postoperative autologous and allogeneic transplantation of trachea, tracheal grafts in the airway epithelium of normal epithelial marker E-cadherin expression was low, and interstitial a -SMA marker of increased epithelial damage and EMT markers of increased expression of MMP-9 (P0.05).
Experimental conclusion: our experimental results show that in the tracheal transplantation model of mice, the occurrence of OAD is accompanied by the phenomenon of airway epithelial to mesenchymal transition, thus providing a possible target for the treatment of OB.
The third part: rapamycin inhibits the occurrence of EMT in the tracheal grafts in mice and reduces obstructive airway disease
Background: rapamycin (RAPA) as immunosuppressive agents used in the treatment of a variety of solid organ rejection. Rapamycin can improve lung function after lung transplantation, but due to many adverse reactions, the treatment of many patients with BOS lung transplantation patients cannot tolerate rapamycin. In this experiment, we try to study the small dose of rapamycin graft treatment the effect of allogenic trachea of mice.
Methods: mice were treated after orthotopic tracheal transplantation group, rapamycin + cosolvent postoperative groups according to given different doses (experimental group) or only given solvent (vector control group). Each group of recipient mice tracheal grafts were transplanted in twenty-eighth days after surgery, pathology and immunohistochemistry. Western-Blot and flow cytometric assessment.
Results: compared with the experimental group, the carrier according to the airway lumen group in syngenic mice trachea graft stenosis (P0.05), epithelial obvious epithelial to mesenchymal transformation: decreased expression of E-Cadherin (P0.05), increased expression of alpha -SMA and MMP-9, the expression level of fiber cell transplantation and peripheral blood in the trachea the increased (P0.05). In the high dose group RAPA treatment had no significant difference (P0.05), the treatment effect of RAPA cutting are stronger than the small dose (P0.05): there was no significant difference in the expression level of fiber cells in the peripheral blood, but on tracheal transplantation fibroblasts expressing levels were lower than the low dose group.
Experimental conclusion: according to our experimental results, a large dose of rapamycin can reduce the occurrence of OAD in airway by inhibiting the occurrence of EMT, and the important role of EMT in the process of OAD formation.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R766

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