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研究生:柯清勳
研究生(外文):Ching-Shiun Ke
論文名稱:藉由影響致癌基因Aurora-A改善人類膀胱癌治療的可行性評估
論文名稱(外文):The role of Aurora-A in curcumin-treated human bladder cancer T24 cells
指導教授:蘇純立
指導教授(外文):Chun-Li Su
學位類別:碩士
校院名稱:長榮大學
系所名稱:護理學研究所
學門:醫藥衛生學門
學類:護理學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:111
中文關鍵詞:薑黃素Aurora-A激酶膀胱癌細胞蛋白表現Aurora-A小干擾RNA中心體分離
外文關鍵詞:curcuminAurora-A kinasesbladder cancer cellsprotein expressionAurora-A siRNAcentrosome separation
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膀胱癌是泌尿道系統最常見的癌症,以化學療法為主軸,但化學藥物常缺乏選擇性,易造成病人的不適,使得護理人員在照護時面臨許多窘境,因此抗癌藥物的研究已成為首要議題。薑黃素(curcumin)是由薑黃(curcuma)萃取而來,具有抗癌、抗發炎、及抗氧化之特性。本研究目的在於檢測薑黃素是否能透過抑制Aurora-A提高膀胱癌的療效。研究結果顯示,薑黃素明顯抑制人類膀胱癌T24細胞的生長。西方點墨法証實薑黃素在48小時30 ?嵱條件下可以明顯抑制Aurora-A、p-Aurora-A (Thr288)或是p-histone 3 (Ser10)蛋白質的表現。RT-PCR結果顯示,此條件亦可使Aurora-A mRNA的表現下降。在12小時20 ?嵱的薑黃素條件下,Aurora-A啟動子活性即有下降情形。以共軛焦顯微鏡觀察Aurora-A、??-tubulin及??-tubulin,發現經由20或30 ?嵱薑黃素處理48小時後,細胞分別呈現multiple centrosome或monopolar spindle現象,與使用Aurora-A siRNA的結果一致。以流式細胞儀檢測未經同步化處理的細胞,結果顯示在48小時30 ?嵱薑黃素的濃度、或單獨使用Aurora-A siRNA,細胞停留G2/M phase比例明顯增加。將細胞starvation同步化在G0期後,在薑黃素作用12小時,細胞停留在G2/M phase比例明顯增加。將細胞以nocodazole同步化在G2期,細胞明顯停留在G2/M期,比較同時間的西方點墨法,Aurora-A蛋白質也有明顯的下降的情形。此研究結果證實,薑黃素抑制T24細胞生長可能與Aurora-A蛋白質有關。
Bladder cancer is one of the common malignancies worldwide, but the prognosis is not good. Curcumin, an active ingredient extracted from the rhizome of the plant Curcuma longa and its related species, exhibits antioxidant, anti-inflammatory, and anti-cancer activities. Aurora kinase A (Aurora-A), is a member of the protein serine/threonine kinase family. Ectopic overexpression of Aurora-A in mammalian cells induces centrosome amplification, chromosome instability, and oncogenic transformation. Gene amplification and/or overexpression of Aurora-A have been reported in urinary bladder cancer. Our data reveal that curcumin significantly inhibited the growth of human bladder cancer T24 cells. Curcumin also decreased the expression of protein and mRNA as well as promoter activity of Aurora-A in a time- and dose-dependent manner. Monopolar spindle and multiple centrosome were observed in T24 cells treated with 30 ?嵱 curcumin for 48 h demonstrated by confocal microscopy. Significant increase in the percentage of cells at G2/M phase was revealed by flow cytometry when asynchronized cells were cultured with 30?n?嵱 curcumin for 48 h. Under this condition, protein expression of Aurora-A was also significantly inhibited by curcumin. After synchronizing the cells at the beginning of G0/G1 phase by starvation, the percentage of cells arrested at G2/M phase was significantly increased when the cells were treated with 30 ?嵱 curcumin for 12 h. Significant increase in the percentage of cells at G2/M phase was displayed when the cells were cultured with 30 ?嵱 curcumin for 10 h after nocodazole treatment to synchronize the cells at the beginning of G2/M phase. Under this condition, protein expression of Aurora-A was also significantly suppressed by curcumin. All together, curcumin may inhibit Aurora-A protein expression and lead to abnormal mitotic spindles and growth arrest, which imply the chemotherapeutic potential of curcumin on bladder cancer cells through suppression of Aurora-A expression.
目 錄
------------------------------------------------------------------------------- 頁數
第一章 緒論 ------------------------------------------------------------ 1
第一節 膀胱癌 ------------------------------------------------------ 2
一、膀胱癌流行率 ------------------------------------------------ 2
二、台灣本土膀胱癌的分佈情形 ----------------------------- 3
三、膀胱癌的致病機轉 ----------------------------------------- 3
四、膀胱癌的治療 ----------------------------------------------- 4
(一) 手術切除 -------------------------------------------------- 4
(二) 放射性治療 ----------------------------------------------- 5
(三) 化學療法 -------------------------------------------------- 5
(四) 生物療法 -------------------------------------------------- 5
(五) 中藥治療 -------------------------------------------------- 6
第二節 薑黃素 ----------------------------------------------------- 6
一、薑黃素的簡介 ----------------------------------------------- 7
二、薑黃素的研究與利用----------------------------------------- 7
第三節 薑黃素與癌症之相關探討 ----------------------------- 9
第四節 Aurora-A 基因對腫瘤的影響 ------------------------- 10
一、Aurora-A在有絲分裂過程中扮演的角色 ------------- 13
(一) 中心體成熟 --------------------------------------------- 13
(二) 中心體的分離 ------------------------------------------ 16
(三) 進入有絲分裂 ------------------------------------------ 16
二、Aurora-A在癌症發展 ------------------------------------ 17
三、Aurora-A和p53腫瘤抑制因子的關係 ----------------- 17
第二章 材料與方法 ------------------------------------------------ 21
第一節 儀器 ----------------------------------------------------- 21
第二節 實驗方法 ----------------------------------------------- 23
一、薑黃素(curcumin)溶液的配製 ----------------------- 23
二、細胞培養 -------------------------------------------------- 23
三、細胞毒殺試驗----------------------------------------------- 26
四、西方墨點法 ----------------------------------------------- 27
五、抽取RNA ---------------------------------------------------- 31
六、反轉錄聚合酵素連鎖反應 ----------------------------- 32
七、冷光酵素活性分析 -------------------------------------- 35
八、siRNA ---------------------------------------------------- 37
九、細胞週期之分析 ----------------------------------------- 39
十、共軛焦顯微鏡(confocal) ------------------------------- 40
第三章 結果與討論
一、薑黃素抑制人類膀胱癌細胞株T24的生長 ------------ 42
二、薑黃素對人類膀胱癌細胞株T24中Aurora-A的蛋白質
表現量變化之影響 ---------------------------------------- 44
三、薑黃素對人類膀胱癌細胞株T24中Aurora-A的mRNA
表現量之影響 ---------------------------------------------- 51
四、薑黃素對人類膀胱癌細胞株T24中Aurora-A的
transcription activity之影響 ------------------------------- 54
五、使用Aurora-A siRNA對人類膀胱癌細胞株T24中
Aurora-A的蛋白質 ----------------------------------------- 57
六、薑黃素對人類膀胱癌細胞株T24內Aurora-A蛋白質
的分佈情形及蛋白質表現量之變化 -------------------- 57
七、薑黃素治療下,人類膀胱癌細胞株T24之細胞週期
分佈,及細胞週期中Aurora-A所扮演的角色 -------- 75
第四章 結論 ----------------------------------------------------------- 104
參考資料 --------------------------------------------------------------- 106
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