臺灣博碩士論文加值系統

中文摘要
乳腺退化是一個高度複雜且多階段的過程。上皮細胞高度的死亡、乳腺脂肪組織的再發展及乳腺組織的重塑為主要特徵。在乳腺退化過程中，會有顯著白血球及巨細噬胞侵入到管腔中及上皮細胞之間，同時細胞黏著分子也會有戲劇性的改變。CD200是一個黏著分子，屬於免疫球蛋白超家族成員，也是一個免疫抑制分子。我們先前的研究也證實CD200確實存在乳腺肌上皮細胞而且與乳腺上皮細胞的生理階段有密切關係。因此，我們的假說是：乳腺肌上皮細胞CD200確實在正常乳腺退化過程中，對於入侵的白血球及巨噬細胞有一個免疫抑制的影響，以維持適當的免疫反應。為了證實這個假說，我們首先檢視大鼠乳腺退化時期，其免疫分子的演變；然後藉由CD200抗體中和內源性CD200後，嘗試去證實有更劇烈的免疫反應及上皮細胞破壞的證據。我們的結果顯示，正常乳腺退化過程中，免疫細胞族群分佈型態的不同主要是根據該免疫細胞所具有的免疫分子特性及乳腺退化階段而有其特異性。在乳腺退化的前七天期間，具CD45免疫反應細胞主要分佈在上皮細胞間，而且隨著乳腺退化天數的增加而有顯著增加趨勢。但是在單位腺泡內，上皮細胞間具CD45免疫反應細胞的數目反而會隨著乳腺退化天數的增加而有顯著減少的趨勢，同樣的趨勢也表現在活性氧屬的產量及一般白血球抗原的表現。而CD200抗體與CD200作用後，確實對於免疫活性也有某種程度的加強效果；其中活性氧屬產量、免疫細胞族群及免疫分子的表現均有增加趨勢，進而加速乳腺退化現象。因此，我們認為在正常乳腺退化過程中，肌上皮細胞CD200一直扮演免疫抑制的調控角色，使乳腺內免疫反應得以適量的進行。

Abstract
Mammary gland involution is a highly complex multi-step process that is characterized by a high degree of epithelial cell death, redevelopment of the mammary adipose tissue and tissue remodeling. A significant infiltration of leukocytes and macrophages into the lumen and between the mammary epithelial cells is also a crucial process during involution that showed drastically altered expression of adhesion molecules. CD200 is an adhesion molecule belonging to immunoglobulin superfamily and well known as an immunosuppressive molecule. In our previous study, CD200 does exist on the myoepithelia of rat mammary glands and be closely related to the physical stages of the mammary epithelial cells. We hypothesize that myoepithelial CD200 may constitutively have an immunosuppressive influence on infiltrated leukocytes/macrophages during normal mammary involution to maintain an appropriate immune reaction. To verify this assumption, the evolution of immunomolecules during gland involution of rats will initially be examined. The neutralization with CD200 antiserum will then be applied to evidence the rigorous immune reaction and epithelial destruction after CD200 antibody ligation. Our preliminary data demonstrated that immune cells varied in distribution dependent on their immunomolecules and the stage of mammary regression. During the first 7 days mammary involution, CD45+ cells were mainly distributed at the intraepithelial site and increased in packing density with time through which their cell density per acinus and ROS were significantly declined. Antibody against CD200 to some extent reinforced the immune activity that showed an increased expression of ROS, immunomolecules and population of the immune cells and may lead to more advanced involution. The present data therefore suggested a constitutively immunosuppressive influence of myoepithelial cells on the immune cells during normal involution of rat mammary gland.

目錄
頁碼
目錄.......................................................................................................I
表目錄...................................................................................................IV
圖目錄...................................................................................................V
附錄目錄...............................................................................................VII
中文摘要...............................................................................................VIII
英文摘要...............................................................................................X
前言.......................................................................................................1
壹、 乳腺的解剖結構.....................................................................1
貳、 各時期的乳腺發展.................................................................3
參、 在乳腺發展過程中肌上皮細胞的分佈與角色.....................10
肆、 乳腺的生長與細胞黏著分子的關係....................................11
伍、 細胞黏著分子CD200的特性.................................................13
研究目的...............................................................................................17
實驗材料...............................................................................................18
壹、 實驗動物.................................................................................18
貳、 實驗藥品.................................................................................18
參、 免疫組織化學染色抗體.........................................................19
肆、 免疫螢光染色抗體.................................................................19
伍、 其他化學試劑.........................................................................20
陸、 儀器設備.................................................................................21
實驗設計與方法...................................................................................22
壹、 實驗設計一：主要觀察大鼠乳腺在強制退化的第一天、第三天及第七天三個生理時期，具CD45、CR3、CD4、IBA1及Fascin免疫反應細胞的分佈型態及強度.........................................22
貳、 實驗設計二：主要證實在乳腺退化期中，肌上皮細胞CD200
的確具有免疫抑制的功能.....................................................23
參、 冷凍切片之動物犧牲與標本組織取得.................................25
肆、 免疫組織化學染色.................................................................26
伍、 免疫螢光染色..........................................................................27
陸、 型態計量分析..........................................................................28
柒、 西方墨點轉漬法......................................................................28
捌、 活性氧屬測定..........................................................................30
玖、 統計分析..................................................................................31
結果.......................................................................................................32
壹、 正常大鼠在強制退化過程中，乳腺的發展..........................32
一、 結構的改變..........................................................................32
二、 免疫分子的分佈及表現.………………………………….32
三、 ROS 的變化..........................................................................36
貳、 使用CD200抗體中和內源性CD200後，對於強制退化乳腺的影響…………………………………………………………..36
一、 結構的改變.…………………………………………….....36
二、 免疫分子的分佈及表現.……………………………….....36
三、 ROS 的變化..........................................................................38
討論…………………………………………………………………....39
壹、 合理的乳腺退化程度參考指標-單位面積內具CD45免疫反應細胞的總數除以腺泡數的數值……………………………..39
貳、 大鼠在乳腺退化過程中，不同的免疫分子及細胞的分佈型態與特性……………………………………………………......40
參、 在乳腺退化過程中，乳腺內注射CD200抗體確實能夠加劇免疫反應，進而加速乳腺退化………………………………..45
結論…………………………………………………………………....51
參考文獻……………………………………………………………....94



表目錄
頁碼
表一、乳腺強制退化前七天，免疫細胞的動態分佈表…………....53
















圖目錄
頁碼
圖一、強制退化乳腺的組織結構…………………………………....55
圖二、乳腺強制退化的過程中，具CD45免疫反應細胞的分佈…..57
圖三、乳腺強制退化的過程中，具CD45免疫反應細胞分佈型態統計................................................................................................59
圖四、乳腺強制退化的過程中，具CD45免疫反應細胞數目統計分析與免疫分子的表現…………………………………………....61
圖五、乳腺強制退化的過程中，具CD4免疫反應細胞的分佈…....63
圖六、乳腺強制退化的過程中，具CR3免疫反應細胞的分佈…....65
圖七、乳腺強制退化第一天，具IBA1 / CR3免疫反應細胞的分佈……………………………………………………………....67
圖八、乳腺強制退化第三天，具IBA1 / CR3免疫反應細胞的分佈……………………………………………………………....69
圖九、乳腺強制退化第七天，具IBA1 / CR3免疫反應細胞的分佈……………………………………………………………....71
圖十、乳腺強制退化過程中，具Fascin / IBA1免疫反應細胞的分佈……………………………………………………………....73
圖十一、乳腺強制退化過程中，ROS 的變化……………………....75
圖十二、在強制退化過程中，乳腺內注射CD200抗體後的乳腺組織結構…………………………………………………………....77
圖十三、在強制退化過程中，乳腺內注射CD200抗體後，具CD45免疫反應細胞的分佈………………………………………....79
圖十四、在強制退化過程中，乳腺內注射CD200抗體後，具CD45免疫反應細胞分佈型態統計………………………………....81
圖十五、在強制退化過程中，乳腺內注射CD200抗體後，CD45蛋白質的表現…………………………………………………....83
圖十六、在強制退化過程中，乳腺內注射CD200抗體後，具CD4 / CR3免疫反應細胞的分佈……………………………………....85
圖十七、在強制退化過程中，乳腺內注射CD200抗體後，具IBA1 / Fascin 免疫反應細胞的分佈…………………………....87
圖十八、在強制退化過程中，乳腺內注射CD200抗體後，ROS的變化…………………………………………………………....89





附錄目錄
頁碼
附錄一、雌性小鼠乳腺分佈圖……………………………………....91
附錄二、免疫細胞三種分佈型態位置圖……………………………93

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