臺灣博碩士論文加值系統

摘要
自體免疫腦下垂體炎 (Autoimmune hypophysitis, AH) 是一個逐漸受到重視的腦下垂體疾病。這個疾病的典型症狀有頭痛、視覺障礙、內分泌功能缺損。其病理特徵是淋巴細胞 (T與B 細胞) 浸潤腦下垂體。然而目前對自體免疫腦下垂體炎的致病機制了解十分有限。在這個研究中，我們利用小鼠模式去探討浸潤腦下垂體的淋巴細胞是否在進行增殖，而如果答案是肯定的，又是何種細胞正在進行增殖。我們誘發小鼠產生自體免疫腦下垂體炎，以proliferating cell nuclear antigen (PCNA) 抗體，利用免疫組織化學染色，發現發病小鼠的腦下垂體中，有許多免疫細胞在進行增生。相較之下，在只有施打免疫完全佐劑的控制組小鼠中，只有少量的細胞在增生。我們利用雙重免疫組織化學染色，使用PCNA和淋巴細胞標誌物的抗體，發現浸潤腦下垂體的T細胞及B細胞在增生。另外有一些腦下垂體內分泌細胞也在增生，可能是腦下垂體遭受淋巴細胞浸潤破壞，因而產生的自我修復。我們使用流式細胞儀分析，也發現浸潤腦下垂體的T細胞中，有約4.53%的細胞在進行增生。而浸潤腦下垂體的B細胞中，有約2.2%的細胞在進行增生。進一步分析發現約4.6%的CD4+ T細胞在進行增生。而有約8.3%的CD8+ T細胞在進行增生。本研究證明了在小鼠的自體免疫腦下垂體炎，浸潤腦下垂體的免疫細胞會在腦下垂體進行增生，此結果使我們對自體免疫腦下垂體炎的致病機制有更進一步的認識，這個發現也可以解釋細胞毒性藥物 (cytotoxic drugs)，如azathioprine，可以藉由抑制細胞分裂來治療自體免疫腦下垂體炎的原因。

Abstract
Autoimmune hypophysitis (AH) is an increasingly recognized disease of the pituitary gland. AH of typically presents with headache, visual-field defects and hormonal deficiencies. The defining pathological feature of AH is lymphocytic infiltration (T and B cells) in the pituitary, however, current understanding to the pathogenesis of AH is very limited. In this study, we aimed to explore whether pituitary-infiltrating lymphocytes proliferate, and if the answer is positive, what kind of cells proliferate, in a mouse model we have previously established. We induced experimental autoimmune hypophysitis (EAH) in the mouse model and studied the proliferation of the pituitary-infiltrating cells by immunohistochemistry. We found that considerable more cells proliferated in the pituitaries of mice that developed EAH, as demonstrated by immunohistochemical stainings using an antibody against proliferating cell nuclear antigen (PCNA). In contrast, only little proliferative activities were noted in the pituitaries of control mice immunized by adjuvants only. By double immunohistochemical staining against PCNA and lymphocyte markers, we found that a portion of pituitary-infiltrating T cells and B cells proliferated in this mouse model. Proliferating T and B cells scattered within the parenchyma of the pituitaries, suggesting a possible mechanism of sustained inflammation caused by T and B cells in the pituitary. Finally, some pituitary parenchymal cells also proliferated, suggesting a regeneration mechanism to compensate the lost pituitary cells caused by inflammation. We confirmed that 4.53% of pituitary-infiltrating T cells and 2.2% of pituitary-infiltrating B cells proliferated by flow cytometry analysis. 4.6% of pituitary-infiltrating CD4+T cell proliferated and 8.3% of pituitary-infiltrating CD8+ T cells proliferated, as demonstrated by flow cytometry analysis. Here we show that pituitary-infiltrating lymphocytes proliferate in situ in EAH. Our results provide a deeper understanding to the pathogenesis of autoimmune hypophysitis. Furthermore, these finding may explain how some cytotoxic drugs such as azathioprine may treat autoimmune hypophysitis.

目錄
中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
圖表目錄 x
一、前言 1
1.1 腦下垂體的介紹 1
1.2 自體免疫腦下垂體炎 3
1.3 以小鼠模式模擬人類自體免疫腦下垂體炎 6
1.4 免疫細胞的分裂及活化 7
1.5 研究目的與動機 9
二、儀器與試藥 10
2.1 儀器 10
2.2 藥品試劑 11
2.3 緩衝溶液及溶劑 13
2.4 培養基 14
2.5 勝任細胞 (Competent cell) 15
2.6 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 所需試劑 15
2.7 抗體 16
三、實驗方法 17
3.1 自體抗原─小鼠生長激素 (mGH) 的製備 17
3.1.1生產SUMO-mGH 嵌合蛋白 17
3.1.2純化SUMO-mGH嵌合蛋白 18
3.1.3透析並純化出mGH蛋白 20
3.1.4利用十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE)，分析純化出
的mGH 21
3.1.5利用西方墨點法 (Western Blotting) 之技術，確認純化出之蛋白
質為本實驗欲使用之mGH 22
3.2 以小鼠生長激素 (mGH) 誘發小鼠自體免疫腦下垂體炎 23
3.2.1誘發小鼠自體免疫腦下垂體炎 23
3.2.2以組織病理切片觀察經誘發自體免疫腦下垂體炎之小鼠發病程度 24
3.3 利用免疫組織化學染色，研究腦下垂體細胞之增生 25
3.3.1準備組織樣品 25
3.3.2組織脫蠟和復水 25
3.3.3組織的抗原修復 25
3.3.4抑制非專一性結合的抗體 26
3.3.5加入一級抗體 26
3.3.6抑制組織中內源性的過氧化氫酶 26
3.3.7加入二級抗體 26
3.3.8加入Streptavidin-Peroxidase放大抗體訊號 27
3.3.9呈色 27
3.3.10復染 (counterstaining) 和封片 27
3.4 利用雙重免疫組織化學染色，研究浸潤腦下垂體免疫細胞之增生 28
3.4.1 抑制非專一性結合的抗體 28
3.4.2 加入一級抗體 28
3.4.3 抑制組織中內源性的過氧化氫酶 29
3.4.4 加入第一重二級抗體 29
3.4.5 第一個呈色反應 30
3.4.6 加入第二重二級抗體 30
3.4.7 第二個呈色反應 30
3.4.8 封片 30
3.5 利用流式細胞儀分析，誘發小鼠自體免疫腦下垂體炎後，發病的腦下垂
體中，增生之免疫細胞亞群 31
四、結果 34
4.1 自體抗原─小鼠生長激素 (mGH) 的生產及純化 34
4.2 以小鼠生長激素 (mGH) 誘發小鼠自體免疫腦下垂體炎 37
4.3 利用免疫組織化學染色，探討小鼠自體免疫腦下垂體炎中，細胞在腦下垂體
的增生 38
4.4 利用雙重免疫組織化學染色，探討小鼠自體免疫腦下垂體炎中，浸潤腦
下垂體T細胞及B 細胞的增生 39
4.5 利用雙重免疫組織化學染色，探討小鼠自體免疫腦下垂體炎發病中晚期
(免疫後56天)，浸潤腦下垂體T細胞及B細胞的增生 41
4.6 在小鼠自體免疫腦下垂體炎，發病中晚期的小鼠腦下垂體中，有正常腦
下垂體細胞及多核巨細胞進行增生 44
4.7 利用流式細胞儀分析在小鼠自體免疫腦下垂體炎，發病腦下垂體中增生
之免疫細胞亞群 45
五、討論 47
5.1 T細胞及B細胞在發病腦下垂體中增生，導致腦下垂體長期發炎 47
5.2 免疫細胞在其他自體免疫疾病發病的非淋巴器官增生之探討 48
5.3 腦下垂體內分泌細胞增生現象 49
5.4 在誘發自體免疫腦下垂體炎，發病中晚期的小鼠腦下垂體中，發現有多
核巨細胞在進行增生 50
5.5 在誘發小鼠自體免疫腦下垂體炎發病的腦下垂體中，增生之免疫細胞亞
群的討論 50
5.6 細胞毒性藥物─Azathioprine，可以治療自體免疫腦下垂體炎的原因 52
六、結論 53
七、參考文獻 54

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