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研究生: 楊采蓁
論文名稱: 1.鑑定以細胞自噬清除神經母細胞瘤堆積多麩醯胺的小分子化合物 2. 鑑定具有清除肝癌類幹細胞潛力的藥物
指導教授: 方剛
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2014
畢業學年度: 102
語文別: 中文
論文頁數: 78
中文關鍵詞: 細胞自噬神經退化性疾病多麩醯胺酸疾病肝癌類幹細胞鴉膽子夏枯草teroxirone苦蔘
論文種類: 學術論文
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  • 1.多麩醯胺酸疾病多屬於神經退化性疾病,主要原因是因為核苷酸CAG異常重複,讓蛋白質錯誤折疊造成聚集所引起細胞毒性,進一步造成細胞死亡。目前已有報導指出,自噬系統可以透過溶酶體降解突變蛋白、胞器並減輕polyQ聚集所引發的細胞毒性,因此促進細胞自噬也許對於神經退化性疾病的治療能提供一個有效的方向。本研究轉殖不同長度polyQ結合綠色螢光蛋白的基因進入人類神經瘤母細胞建立穩定細胞株作為篩選平台,實驗首先利用螢光染色標定細胞自噬相關標記,篩選出能誘導細胞自噬並且不會影響細胞的存活的化合物。再透過螢光顯微鏡觀察藥物是否可以加強清除蛋白堆積物。此外在西方轉漬法,確定藥物能增加細胞自噬的相關標記蛋白。最後利用自噬抑制劑抑制細胞自噬進而降低清除polyQ聚集蛋白的能力,以證明藥物是透過促進自噬方式清除毒性蛋白堆積物。本研究所篩選的藥物,可作為治療神經退化性疾病的潛力藥物的發展,未來將更進一步確認細胞自噬如何清除細胞polyQ 堆積的相關機制。

    2.肝癌是全球排名第三與癌症死亡有關的疾病,雖然手術切除可以提高存活率,但是在術後常發現還是有癌細胞轉移及擴散的現象。癌症幹細胞具有自我更新以及分化能力,認為是造成癌症轉移、復發,對於化療、放射性療法產生抗藥性的主要原因,由於腫瘤細胞亞群可以抵抗藥物毒性導致在癌症病患體內要徹底清除癌細胞是一大挑戰。本論文使用肝細胞株Huh7 (mutant p53),HepG2 (wild Type p53)和Hep3B (p53 null)分別處理teroxirone以及各種中藥,觀察細胞型態。實驗首先要培養肝癌類幹細胞,將肝癌細胞培養在不含血清但含有特定生長因子的培養基(DMEM/F12)裡面含鹼性成纖維細胞生長因子(basic fibroblast growth factor; bFGF),表皮生長因子(epidermal growth factor; EGF)以及B27,培養7天後,將培養出tumor sphere,實驗以不同濃度的teroxirone以及中藥處理進一步利用顯微鏡觀察spheroid的生成和大小,並鑑定相關蛋白質變化。目前本研究發現這些藥物會有不同程度降低tumor sphere的體積及數目,會進一步會利用癌症標誌物分離肝癌spheroid的細胞,未來將具有癌症幹細胞特徵的細胞使用萃取緩衝液萃取出蛋白,再利用西方點墨法觀察幹細胞相關標誌物以及可能的細胞凋亡機制,期望本研究可以利用不同藥物以降低肝癌類幹細胞生長以及有效抑制腫瘤的增生。

    第一部分 目錄 中文摘要————————————————3 英文摘要————————————————4 壹、文獻回顧——————————————5 貳、研究背景——————————————10 參、研究目標——————————————12 肆、研究材料與方法———————————12 伍、研究結果——————————————19 陸、討論 ———————————————24 柒、參考文獻——————————————27 捌、圖示————————————————32 第二部分 目錄 中文摘要———————————————42 英文摘要———————————————43 壹、文獻回顧—————————————45 貳、研究背景—————————————50 參、研究目標—————————————55 肆、研究材料與方法——————————55 伍、研究結果—————————————59 陸、討論 ——————————————61 柒、參考文獻—————————————63 捌、圖示———————————————68

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