臺灣博碩士論文加值系統

發炎所引起的肝臟疾病，無論是急性或慢性肝炎，一直是全人類的健康問題，而慢性肝炎更是與肝細胞癌的形成有密切相關。因此長久以來科學家即不斷發展實驗動物模式來研究肝臟特異性的免疫調節機制，目前由Concanavalin A ( Con A )所誘發的小鼠肝炎被認為和人類自體免疫性肝炎所造成的反應是類似的。過去已知的研究中，因自體免疫造成肝炎的嚴重程度在兩性中有顯著的差異。我們要利用Con A誘發肝炎的模式，瞭解性荷爾蒙在這個肝炎模式中扮演的角色。Con A是一種源自植物的lectin，會特異性與T細胞表面醣蛋白上的特異糖分子，如T細胞受體或CD3分子結合，經由這些分子刺激T細胞活化，它是一種能使T淋巴細胞活化增生的裂殖原。當不同品系的小鼠以尾靜脈方式給予Con A ( 10 mg/kg )後，在24小時內，藉由測量血清中轉胺酵素可以發現小鼠產生急性肝炎，並會持續至36到48小時左右。然而Con A誘發的肝炎會隨著不同品系與性別的小鼠，其嚴重度有明顯差異。C57BL/6與BALB/c品系小鼠對Con A所誘發之肝炎敏感度較A/J品系小鼠高，而雌鼠則比公鼠產生更嚴重的肝炎。Con A誘發之肝炎會造成大量T淋巴細胞浸潤聚集到肝臟中，其中以CD4+ T淋巴細胞最為顯著，而從流式細胞儀的偵測，發現經誘導後的雌鼠，其浸潤的CD4+與CD8+ T細胞遠較公鼠多。為了更進一步探討性荷爾蒙在此肝炎模式的角色，我們以外科手術去除掉成熟雌鼠之卵巢二週後，再給予Con A刺激，發現卵巢去除後的雌鼠，血清中的轉胺酵素明顯降低，程度近似公鼠，而以雌性荷爾蒙再補充的雌鼠即會回復肝炎的程度，其CD4+與CD8+ T細胞數量較未給予雌激素補充的雌鼠多，並且有顯著的統計上差異。測量一些細胞激素，如IL-4、IL-6、IL-10、IL-12、TNF-α、IFN-γ，我們發現它們的量在正常雌鼠與卵巢切除後再補充雌性激素之雌鼠皆高於公鼠以及只有卵巢去除的雌鼠。另外自然殺手性T細胞的數量，在未受過任何處理的公母鼠中，即具有差異，在給予Con A後，其細胞數目也有變化，顯示自然殺手性T細胞也參與在Con A誘發之肝炎模式中。由以上的結果顯示，性荷爾蒙會參與調節Con A誘發的小鼠肝炎，但雌激素是如何作用及調節細胞激素與免疫細胞則須更進一步的研究。

An inflammation-mediated liver disease, both acute and chronic hepatitis, represents a worldwide health problem in human beings, and chronic hepatitis is associated strongly with the development of hepatocellular carcinoma. Experimental animal models have been used for a long time to study liver-specific immune regulation. Concanavalin A ( Con A ) — induced hepatitis in mice is thought to mimic the autoimmune hepatitis in human. The degree of autoimmune hepatitis is known to be different between genders — female is severe than male. In this study, we used the Con A-induced hepatitis model to investigate the role of sex hormones in autoimmune hepatitis. Con A is a lectin from plant, it binds specifically to certain sugar residues on T cell surface glycoproteins such as TCR or CD3 molecules and then stimulates the T cells to proliferate. It is known as a mitogen for T lymphocytes. When different strains of mice were given a single intravenous injection of Con A (10 mg/kg ). The acute hepatic injury assessed by increased plasma transaminase levels developed within 24 hours and lasted until 36 or 48 hours. The severities of liver injury were different among different strains and gender. C57BL/6 and BALB/c mice were more sensitive to Con A-induced hepatitis than A/J mice. The Con A-induced hepatitis is more severe in female mice than in male mice. The number of T lymphocytes in the liver was increased after Con A injection, especially the CD4+ T cells. There were more infiltrated CD4+ and CD8+ T cells in female than in male mice. We are interested in the hormonal regulation of this hepatitis model. Female mice were ovariectomized for two weeks, then challenged with Con A. The plasma transaminase levels of these levels of these ovariectomized mice were reduced to those of male mice. However after the estrogen supplement in these ovariectomied mice, the severity of Con A-induced hepatitis could be restored to the level of female mice. The CD4+ and CD8+ infiltrating T cells were increased significantly in estradiol supplemented ovariectomied mice compared to ovariectomied non-supplemented mice. The cytokines of IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ in liver after Con A stimulation were found higher in female or estradiol supplemented ovariectomized mice than male or ovariectomized mice. Furthermore, the natural killer T (NKT) cells in liver were different in different genders of mice. After Con A stimulation, the number of these NKT cells changed as well. This suggests that NKT may also be affected by estradiol in this Con A-induced hepatitis.

目錄
授權書
考試合格證明
誌謝
中文摘要 I
英文摘要 III
目錄 V
表目錄 VII
圖目錄 VIII
符號與縮寫 IX
主文
緒論 1
材料與方法 10
一、方法
A、動物實驗 10
B、免疫細胞浸潤分析 11
C、細胞激素的偵測與定量 15
D、以ovariectomy手術來探討荷爾蒙在肝炎模式的角色 21
二、材料 23
A、藥品與培養液 23
B、其他試劑 27
C、耗材 28
D、儀器 28
實驗結果
I.Concanavalin A誘發小鼠急性肝炎模式 30
II.Concanavalin誘發小鼠肝炎後的組織病理變化 30
III.Con A導致細胞激素的釋放與作用 31
IV.探討公母鼠在Con A誘發急性肝炎上的差異 32
V.雌性荷爾蒙對Con A引發急性肝炎的影響 34
VI.NK T細胞在Con A引發肝炎中的變化 36
討論 38
參考文獻 45
表 54
圖 59

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