臺灣博碩士論文加值系統

本研究的目的是希望找到一個移植模式較簡單、免疫抑制時間短，又能誘發出肢體移植後移植物抗宿主病（GVHD）的動物模式，以方便進一步對肢體移植後GVHD的研究。我們在移植模式方面採用了骨骼移植模式、骨骼及淋巴結移植模式、骨骼肌肉皮膚移植模式與完整肢體移植模式四種不同複雜程度的移植模式。在移植後免疫抑制計畫方面，我們使用cyclosporine(長時間：5mg/kg 60天，短時間：5mg/kg 30天)與FK506（高劑量：0.5mg/kg，低劑量：0.1mg/kg）同時併用leflunomide（10mg/kg）。停藥後觀察是否出現臨床GVHD的症狀，同時監控移植物被排斥的情形。實驗動物犧牲後，進行病理學上的檢查並利用免疫組織化學染色的方式觀察接受者脾臟內巨噬細胞(使用ED1抗體)與自然殺手細胞（使用NKR-P1抗體）的活性與數量變化，然後將上述所得肢體移植後GVHD的特性和文獻中骨髓移植或親代移植給第一子代模式後GVHD相互比較，並評估各種模式的可行性。結果發現，在臨床上僅有完整肢體移植模式能夠誘發出臨床GVHD，6隻實驗動物中有2隻出現臨床症狀。臨床症狀包括體重急速下降與呼吸急促。組織病變則以肺臟、腸道與淋巴系統（胸腺，腸繫膜淋巴結）為主。不過在部分使用骨骼肌肉皮膚移植模式，高劑量FK506 與leflunomide進行免疫抑制的實驗動物雖然在臨床上沒有出現症狀，但在病理學檢查方面可以發現疑似GVHD的病變。在移植物被排斥方面：完整肢體移植模式較骨骼肌肉皮膚移植模式容易被排斥，而高劑量的FK506則能夠提供移植物較好的保護。而巨噬細胞和自然殺手細胞的變化和骨髓移植後的GVHD相似，巨噬細胞活性上升；自然殺手細胞的活性則在GVHD早期被刺激升高，晚期呈現抑制的情形。

The purpose of this study is to establish a simple surgical model of limb allotransplantation in order to study the nature of graft versus host disease. Four transplantation models were developed, which included the bone transplant model, bone and lymph node transplant model, Osteo-myo-cutaneous (OMC) transplant model and the traditional full-limb transplant model. Brown Norway(BN) to Lewis(LEW) rats combination was used in the study. Leflunomide at the dose of 10mg/kg with cyclosporine (short term:30 days, Long term: 60 days) or FK506 (low-dose: 0.1 mg/kg, high-dose: 0.5 mg/kg) /kg) were given as immunosuppresants after transplantation. After stopped giving the immunosuppression agents, clinical sign of GVHD or rejection of the grafts were observed and histologically examined using ED1 and NKR-P1 monoclonal antibodies in order to investigate the macrophage and NK cell activity during GVHD. Finally, we compared the GVHD after limb allotransplantation with the GVHD after clinical bone marrow transplantation and P to F1 animal model in the references. As a result, only the traditional limb transplantation model developed GVHD, two of six rats showed clinical signs including sudden weight loss, poor spirit, ocular and nasal discharge, and dyspnea. Lung and lymphoid tissues (thymus and mesenteric lymph nodes) were the major target organs in pathology, which was different from GVHD after BMT. However, some rats in the OMC model, with high dose of FK506 showed the histological but not clinical evidence of GVHD. In the observation of graft rejection, the traditional limb grafts were more easily rejected than the OMC model. And high dose FK506 combined with leflunomide gave more protection than other immunosupression protocol. The macrophage activity was increased during GVHD, and the NK cell activity was increased at the early phase but decreased at the late phase of GVHD, these are similar to the changes during GVHD after BMT.

表次………………………………………………………………………IV
圖次……………………………………………………………………….V
縮寫符號………………………………………………………………….X
中文摘要……………………………………………………………….XII
英文摘要………………………………………………………………XIII
第一章 緒言………………………………………………………………1
第二章 文獻探討…………………………………………………….3
第一節 移植物抗宿主病的定義，分類與機制…………………….3
一. 定義………………………………………………………………3
二. 分類………………………………………………………………3
三. 機制………………………………………………………………6
第二節 移植物抗宿主病的臨床特性……………………………….14
一. 臨床好發情形……………………………………………………14
二. 臨床症狀…………………………………………………………15
三. 臨床診斷與分級…………………………………………………18
四. 臨床預防與治療…………………………………………………21
第三節 移植物抗宿主病的動物研究模式………………………….35
一. 實驗動物品系配對的選擇………………………………………35
二. 淋巴球或骨髓細胞移植模式……………………………………41
三. 小腸移植模式……………………………………………………42
四. 複合組織移植模式………………………………………………43
第四節 移植物抗宿主病的病理特性……………………………….46
一. 皮膚………………………………………………………………47
二. 肝臟………………………………………………………………49
三. 消化道……………………………………………………………51
四. 淋巴系統…………………………………………………………53
五. 其他………………………………………………………………55
第五節 移植物抗宿主病的偵測方法……………………………….58
一. 傳統偵測方法……………………………………………………58
二. 利用細胞激素的變化偵測………………………………………59
三. 利用細胞功能性與組成的變化偵測……………………………61
四. 利用細胞交移的變化偵測………………………………………62
五. 其他發展中方法…………………………………………………63
第三章 實驗材料………………………………………………………64
第四章 實驗方法………………………………………………………72
第一節 實驗分組………………………………………………………72
第二節 移植模式………………………………………………………75
第三節 免疫抑制劑的配製與投與……………………………………90
第四節 術後觀察與評估………………………………………………91
第五章 結果……………………………………………………………102
第一節 臨床觀察與記錄………………………………………………102
第二節 體重變化記錄…………………………………………………116
第三節 病理學檢查……………………………………………………129
第四節 免疫組織化學染色……………………………………………147
第六章 討論……………………………………………………………152
第一節 異體肢體移植後GVHD和骨髓移植及P-F1移植後GVHD之比較152
第二節 本研究中不同移植模式間之比較……………………………160
第三節 不同免疫抑制劑FK506與cyclosporine之比較……………169
第四節 巨噬細胞和自然殺手細胞和異體肢體移植後GVHD之關連性173
第五節 實驗方法方面…………………………………………………176
第七章 參考文獻………………………………………………………177

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