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研究生:劉銘聖
研究生(外文):Ming-Sheng Liu
論文名稱:Nuclear Corepressor-2以外基因機制調控Fibronectin在乳腺分枝型態形成所扮演的角色
論文名稱(外文):The Role of Nuclear Corepressor-2-mediated Epigenetic Regulation on Fibronectin Expression in MammaryBranching Morphogenesis
指導教授:高振益高振益引用關係
指導教授(外文):Jung-Yie Kao
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:43
中文關鍵詞:乳腺外基因Nuclear receptor co-repressor 2 (N-CoR2)三維組織培養模式Fibronectin終端末芽
外文關鍵詞:Mammary glandsEpigeneticsNuclear receptor co-repressor 2Three-dimensional mammary morphogenesis modelsFibronectinTerminal end buds
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乳腺(Mammary glands)的分枝形態形成(Branching morphogenesis) 需要細胞與細胞間以及細胞與體外基質 (Extracellular matrix) 間交互作用的變化,然而造成這些變化的分子機轉仍然不完全清楚。我們在此運用免疫組織染色法和生物工程學的技術發現一外基因型 (Epigenetic) 調控蛋白Nuclear receptor co-repressor 2 (N-CoR2) 在乳腺的分枝形態形成過程中扮演著極為重要的角色。在正常人類乳腺上皮,N-CoR2的表現量具有明顯的局部差異性 (Regional heterogeneity),其在乳腺腺泡 (Mammary acini) 中的表現量高於鄰近的腺管 (Ductules)。我們進一步利用三維組織培養模式 (Three-dimensional tissue culture model) 模擬乳腺形態形成的過程,據此發現經由RNA干擾調控降低內生性N-CoR2的乳腺上皮細胞所形成的腺胞週邊細胞外蛋白Fibronectin 的表現非常明顯的上升,此一變化會伴隨著上皮細胞鈣黏蛋白 (E-cadherin) 的表現下降以及腺胞分支 (Branching) 現象的出現。我們進一步分析小鼠乳腺發育過程期間中N-COR2及Fibronectin的表達情形,我們發現在乳腺發展中的終端末芽 (Terminal end buds (TEBs)) 中N-CoR2主要由位在最外層中的腔道上皮細胞 (Luminal epithelial cells) 所表現,且重要的是,其表現會突然的在分枝點 (Bifurcation site) 位置下降。此一嶄新的發現顯示N-COR2的表現確實會隨著特定區域而改變,伴隨著此一變化的是在分枝點位置中腔道表皮細胞會明顯與其周邊累積Fibronectin,此與體外實驗研究結果一致。總結來說,我們發現到透過N-CoR2相關的外基因調控影響乳腺分枝形態形成的嶄新機制。

Branching morphogenesis of mammary glands requires site-specific alterations in cell-cell and cell-matrix adhesions; However, the molecular mechanisms underlying this developmental process remain less understood. Using immunohistochemical and bioengineering approaches, we show here that nuclear receptor co-repressor 2 (N-CoR2) plays a crucial role in regulating mammary branching morphogenesis. In normal human breast epithelium, N-CoR2 displays a considerable inter-regional expressional heterogeneity with its levels higher in mammary acini than the adjacent ductules. Using three-dimensional mammary morphogenesis models, we showed that RNA-interference-mediated knockdown of endogenous N-CoR2 in mammary acini led to a dramatic upregulation of the extracellular protein fibronectin (FN) accompanied with reduced E-cadherin expression, which were functionally linked to the initiation of branching. In line with these in vitro findings, we further provided compelling evidence for the role of N-CoR2-mediated regulation on FN expression during the developmental process of mouse mammary glands. Specifically, N-CoR2 is predominantly expressed in the luminal epithelial cells of the outermost layer of the developing terminal end buds (TEBs) and its expression abruptly declines at the bifurcation sites. Coincidental with the site-specific changes of N-CoR2 expression, the luminal epithelial cells accumulate pericellular FN at the bifurcation sites. Together, our findings suggest a novel paradigm of an epigenetic regulation for mammary branching morphogenesis mediated by N-CoR2.

表次....................................................iii

圖次.....................................................iv

附圖次....................................................v

縮寫表...................................................vi

一、 緒論.............................................1

二、 文獻探討.........................................3
一、 乳腺發育生物學..................................3
二、 外基因與腫瘤發生................................5
三、 外基因與腫瘤癌化的過程..........................6
四、 N-CoR2非僅是nuclear receptor corepressor........7

三、 材料與方法........................................9
一、 試劑和抗體.....................................9
二、 儀器設備......................................10
三、 細胞培養......................................10
四、 動物實驗......................................11
五、 組織晶片......................................11
六、 RNA萃取與純化.................................12
七、 即時定量聚合酶連鎖反應........................12
八、 西方點墨......................................13
九、 三維空間細胞培養 (3D Culture) 模式建立........13
十、 免疫螢光染色..................................14
十一、 乳腺摘除及石蠟包埋切片........................14
十二、 免疫組織染色方法..............................15
十三、 免疫螢光分析和分支 (Branching) 或分散的定量...15
十四、 數據分析......................................15

四、 結果.............................................16
一、 在人類乳腺中對於N-CoR2和其它相關基因的免疫組織化
學分析........................................16
二、 N-CoR2調節細胞貼附著 (Cell Adhesion) 與移動蛋
白 (Cell Motility) 的表現.....................16
三、 In Vitro中N-CoR2和FN調節乳腺的分枝形態形成
(Branching Morphogenesis) ....................16
四、 In Vivo中乳腺分枝形態形成期間會造成N-CoR2在特定
位置的調節下降................................17

五、 討論.............................................19

六、 總結及N-CoR2未來研究方向.........................20

七、 參考文獻.........................................22



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