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研究生:蔡健婷
研究生(外文):Chien-ting Tsai
論文名稱:ACSL在癌細胞中所扮演的角色
論文名稱(外文):The Novel Role of Acyl-CoA Synthetase Long-chain Family Members in Cancer
指導教授:賴明德賴明德引用關係
指導教授(外文):Ming-Derg Lai
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:88
中文關鍵詞:脂質肝癌內質網壓力
外文關鍵詞:cancerendoplasmic reticulum stressfatty acidAcyl-CoA synthetase long-chain familysiRNA
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在真核細胞中,維持細胞的�琠w與脂質的製造最主要的胞器是內質網(Endoplasmic Reticulum;ER),當蛋白質在製造過程中無法正常的摺疊(folded)或發生構型異常(misfloded),就會累積在內質網中造成內質網壓力,進而啟動內質網上的三個感應器(sencer),分別為PKR-related ER kinase(PERK), inositol requiring enzyme-1(IRE1), 以及activating transcription factor 6(ATF6)。許多原因都會引起內質網壓力,像是B型肝炎病毒或是C 型肝炎病毒的感染、酒精的濫用等等,而在這些情況下都會造成罹患脂肪肝的比例增加。此外,在數十種惡性腫瘤,像是肝癌( hepatocellular carcinoma ;HCC )中,都有伴隨內質網壓力升高的情形,然而目前對於內質網壓力是如何引起脂質累積的機制仍需進一步釐清。脂質合成酶(Fatty acid synthase;FAS)是脂質合成過程中的重要酵素,在本實驗室之前的研究發現脂質合成酶的抑制劑淺藍菌素(Cerulenin),無法成功抑制內質網壓力造成的脂質累積情形。 而在合成脂質時,細胞除了重新合成的路徑(de novo pathway)外,也會利用外源性的游離脂肪酸併入三酸甘油酯(triacylglycerol;TG)中。在本篇研究中,我們著重於研究長鏈型醯基輔酵素A 合成酵素(Aacyl-CoA synthetase long-chain;ACSL),它是脂肪酸要形成TG 過程中,第一個作用的酵素,此酵素主要可將長鏈脂肪酸催化為醯基輔酵素A 硫酯(acyl-CoA thioesters),在進入生合成或代謝的路徑。ACSL 具有5 個成員,分別是ACSL1、ACSL3、ACSL4、ACSL5以及ACSL6。除了ACSL6 以外,其他四個成員在肝細胞當中都有表現。我們接著利用ACSL1、ACSL3 和ACSL 4 的共同抑制劑Triacsin C,發現可以成功抑制掉內質網壓力造成的脂質累積現象。其中會受到Triacsin C抑制的ACSL3 可以受到內質網壓力的調控而活化。此活化現象可能是透過Eukaryotic translation initiation factor 2 alpha subunite(eIF-2alpha)或是Apoptosis signal-regulating kinase 1 (ASK1)複合體的調控。我們進一步利用核醣核酸干擾技術(RNAi)抑制Huh7 細胞中的ACSL3,也可以成功抑制掉內質網壓力造成的脂質累積現象。並發現在細胞中,ACSL3 的缺乏抑制了細胞的爬行能力,並且生長速率稍慢但沒有顯著的差異。而在利用流式細胞儀Flow Cytometry 分析細胞週期發現,抑制ACSL3 似乎可以令Huh7 對於抗癌藥物5-氟尿嘧啶(5-Fluorouracil;5-FU)的敏感度。在未來我們將會進一步觀察ACSL3 siRNA 是否可以作為新的癌症治療分子。
The endoplasmic reticulum (ER) is a major organelle maintaining cellular homeostasis. Accumulation of unfolded /misfolded proteins in the ER lumen induces ER stress which is mediated by three sensors, PKR-related ER kinase (PERK), inositol requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). ER stress is induced in many situations such as HBV or HCV infection, alcohol consumption, and the rate of fatty liver is elevated under these diseases. Moreover, ER stress is induced in at least ten different cancers such as hepatocellular carcinoma (HCC). However, the signaling pathways and mechanisms linking ER stress to lipid accumulation are not well characterized. Fatty acid synthase (FAS) is a key enzyme in de novo lipogenesis pathway. Our lab’s previous study has shown that the FAS inhibitor, Cerulenin, could not block the lipid accumulation under ER stress condition. Besides de novo pathway, exogenous free fatty acids could also incorporation into triacylglycerol (TAG). In this study, we focus on acyl-CoA synthetase long-chain (ACSL) family, which catalyzed the first step in fatty acid metabolism by converting long-chain fatty acid into acyl-CoA thioesters and entered both anabolic and catabolic pathways. The family of mammalian ACSLs consisted of five members, ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6. All members could be detected in liver except ACSL6. We used Triacsin C, an inhibitor of recombinant ACSL1, 3, and 4 to block the lipid accumulation under ER stress successfully. Among these members, ACSL3 could be induced by ER stress. This activation was probably mediated through eIF-2alpha and ASK1 complex activation. But the detail mechanism of ACSL family activation remained to be defined. Therefore, the ACSL3 knockdown stable clones were established and the lipid accumulation under ER stress was blocked. We further investigated the role of ACSL3 in tumerogenesis under ER stress. We used MTT assay and boyden chamber to detect cell growth and migration. The growth rates had no significant change but migration activity of ACSL3 siRNA stable clone is lower than parental cell. We use flow cytometry to analyze cell cycle, and found that ACSL3 siRNA might increase sensitivity of Huh7 cells to 5 Fluorouracil. In the future, we would test whether ACSL3 siRNA can be a therapeutic agent.
緒論
一、腫瘤細胞和脂質的關聯性 1
二、內質網壓力和脂質的關聯性 2
三、ACSL 在脂質合成中扮演的角色 3
四、研究目標與策略 6
材料與方法
一、 細胞培養與藥物處理 8
二、 質體製備 13
三、 抽取total RNA 16
四、 反轉錄聚合酶連鎖反應 20
五、 構築pHsU6-human-ACSL3-siRNA 21
六、 基因殖入轉染(Transfection)與細胞株挑選 26
七、 油紅染色 27
八、 膽固醇染色 28
九、 細胞生長分析計算 29
十、 細胞移動分析 29
十一、 西方墨點法 31
十二、 流式細胞儀分析細胞週期 37
十三、 脂質萃取 39
十四、 薄層層析法分析脂質 40
十五、 高效液相層析儀分析脂質 41
結果
一、 內質網壓力(ER stress)可調控ACSL 的表現 43
二、 抑制ACSL3可以抑制內質網壓力造成的脂質累積情形 43
三、 內質網壓力透過eIF-2α 及ASK1 複合體而調控ACSL3 45
四、 降低細胞內ACSL3的表現會抑制細胞爬行 45
五、 抑制ACSL3可增加Huh7細胞對於5-Fluorouracil 的敏感度 47
六、 分析內質網壓力下的脂質組成的變化 47
討論 49
結論 54
參考文獻 55
圖 59
附錄 78
自述 88
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