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研究生:陳彥孜
研究生(外文):Yan-Tzu Chen
論文名稱:分泌性蛋白Lcn2對初級小鼠子宮內膜上皮細胞生理機制的影響
論文名稱(外文):The Effects of Secretory Lcn2 Protein on Mouse Primary Endometrial Epithelium.
指導教授:朱善德
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
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:57
中文關鍵詞:Lcn2小鼠子宮內膜上皮細胞表皮細胞轉換至間質細胞細胞角蛋白纖維連接蛋白
外文關鍵詞:Lcn2mouse endometrial epitheliumepithelial to mesenchymal transition (EMT)cytokeratinfibronectin
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Lcn2是疏水性分子結合蛋白家族中的一員,在哺乳類的系統中屬於分泌性蛋白,可做自泌或旁泌的生理反應,已知Lcn2為一種急性反應蛋白(actue phase protein),在細胞受到多種不同環境壓力時會被誘導分泌。由於在細胞遭受環境壓力時可能會由表皮細胞轉換成間質細胞(Epithelial-mesenchymal transition, EMT)的現象,許多文獻也指出Lcn2與EMT之間可能有互動關係。為了解Lcn2是否參與在生殖系統中EMT過程,因此根據Glasser等人的方法,建立初級培養小鼠子宮內膜上皮細胞培養系統,並以cytokeratin抗體鑑定之。採用長時間營養缺乏的環境壓力培養條件來誘導Lcn2蛋白的產生,探討Lcn2是否會使小鼠子宮內膜上皮細胞發生EMT,以及誘發EMT對子宮內膜上皮細胞的生理現象是否造成影響。實驗結果顯示在環境的壓力下可以誘導初級小鼠子宮內膜上皮細胞表現Lcn2蛋白,且Lcn2蛋白可以促進初級小鼠子宮內膜上皮細胞轉化成間質細胞,造成上皮細胞標誌-細胞角蛋白(cytokeratin)表現量減少,間質細胞標誌-纖維連接蛋白(fibronectin)表現量增加,導致細胞由聚集狀態轉變為紡錘狀及分散分布,此一變化可能會增加細胞的移動能力和侵襲能力,推測Lcn2可能參與子宮內膜病變的生成。

Lcn2, a member of the lipocalin family, is as a secretory protein in mammalian system as well as has autocrine and paracrine physical functions. A well known, Lcn2 is an acute phase protein and is induced by stress conditions in cells. Owing to the stress, epithelial to mesenchymal transition (EMT) could occur and the substantial evidences encourage us to figure out the relationship between Lcn2 and EMT in primary endometrial epithelium cell. To establish primary culture system of mouse endometrial epithelium cell is our matter of immediate urgency. According to the method of Glasser et al, after trypsin digestion, we haversted primary endometrial epithelium cells and the cells have been identified with cytokeratin antibody. Using nutrition deprivation as a stress condition, we could induce highly Lcn2 expression in this endometrial epithelium cell. To elucidate whether Lcn2 triggers the EMT in endometrial epithelium cell then initiates the influence of the cell function in reproductive system is our goal. As the Lcn2 expression in high level, the cell morphology would change from round-up to spindle-like then the cell dispersion would happen. Meanwhile, double immunostaining showed cytokeratin expression was diminished and fibronectin expression was increased, and an additive effects on cell migration and cell invasion. These results demonstrate that Lcn2 could be induced by environmental stress and trigger the EMT in primary mouse endometrial epithelium cell. These data shield some light on the participation of Lcn2 in the pathologies of endometrial system.

摘要................................ i
Abstract.............................. ii
縮寫表............................... iii
目錄................................ v
圖表目錄............................. vii
前言................................ 1
材料與方法............................ 9
小鼠子宮內膜上皮細胞初級培養....................... 9
免疫螢光染色......................... 10
細胞總體RNA萃取.......................... 11
反轉錄反應........................... 11
聚合酵素連鎖反應........................ 12
洋菜膠電泳........................... 13
即時定量聚合酵素連鎖反應.................... 14
細胞蛋白質萃取......................... 14
蛋白質定量............................. 15
十二指硫酸鈉-聚丙烯安膠體電泳................. 15
西方墨點............................ 16
創傷癒合分析.......................... 17
細胞移動力分析........................ 17
細胞侵襲力分析.. ....................... 18
結果................................ 19
建立子宮內膜上皮細胞初級培養..................... 19
子宮內膜上皮細胞之生長情形......................... 19
子宮內膜上皮細胞於環境壓力下Lcn2表現量.............. 20
長期培養子宮內膜上皮細胞的細胞形態變化............. 21
子宮內膜上皮細胞中EMT細胞標誌的變化.............. 21
Lcn2對細胞EMT的影響.......................... 22
Lcn2對於細胞移動(migration)能力及侵襲(invasion)能力的影響.................. 23
討論................................ 25
參考文獻.............................. 48
附錄................................ 57
附錄一 RT-PCR primer 序列.................... 57

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