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研究生:吳家如
研究生(外文):Jia-ru Wu
論文名稱:Snail在TPA造成肝癌細胞生長抑制、上皮-間質轉型及移動之多重性調節作用
論文名稱(外文):Multiple Roles of Snail in Regulation for TPA-Triggered Growth Inhibition, Epithelial-Mesenchymal Transition and Migration of HepG2 Cells
指導教授:吳文陞
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:94
語文別:中文
論文頁數:63
中文關鍵詞:Snail
外文關鍵詞:Snail
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我們探討佛波醇酯腫瘤促進劑TPA (12-O-tetradecanoyl-phorbol 13-acetate)造成肝癌細胞株HepG2生長抑制、EMT (epithelial-mesenchymal transitions)以及細胞移動的轉錄機制。根據過去的報導指出, 轉錄因子Snail會抑制細胞間黏附複合體中E-cadherin的表達而導致EMT的發生與細胞的移動;另一方面, Snail亦可透過降低Cyclin D1的表達而使得細胞週期產生停滯的現象。
在先前的實驗中, 我們發現TPA可引發HepG2細胞中Snail的表達。接著以antisense Snail oligodeoxynucleotides (AS Snail ODNs)轉染至細胞後則發現可避免掉TPA對HepG2引發的作用, 包括生長抑制 (細胞週期靜止在G1 phase), EMT以及細胞的移動。此外, 在AS Snail ODN轉染至HepG2後也可避免TPA誘發Cyclin依賴性激酶 (CDK)抑制蛋白p15INK4b基因的表達以及抑制E-cadherin和Cyclin D1基因的表達。
除了用AS Snail ODNs抑制TPA引發的Snail表現研究Snail是否為TPA造成HepG2細胞移動與生長抑制現象所需之蛋白之外, 我們亦建立穩定表達Snail的HepG2細胞, 於細胞移動及生長抑制兩方面做觀察, 結果發現單獨Snail的表現雖不足以抑制E-cadherin的表達但卻可促進細胞移動性的增加, 另外, 在Snail單獨的表現下Cyclin D1的表現不受影響, 但是卻能增加p15INK4b基因的表達並促進細胞在G1階段的比例。
總結我們的實驗結果顯示,轉錄因子Snail基因之表現為TPA抑制E-cadherin基因表達而造成HepG2細胞EMT及移動以及誘發CDK抑制者p15INK4b基因的表達以及抑制Cyclin D1的表達而導致HepG2生長停滯所必須。在Snail overexpressing HepG2的細胞中只有p15 INK4b如我們預期有表現量的提高, 而Cyclin D1及E-cahedrin則意外的沒有被抑制甚至E-cahedrin的表現不降反升。這個不一致的現象反映著snail調節基因表現的複雜性。未來, 我們將試著研究一些與EMT和細胞移動相關的基因, 如Fibronectin, Integrin, MMPs等, 以瞭解在Snail overexpressing HepG2細胞中 Snail是藉由調節哪些基因而造成這些作用。
We are investigating the transcriptional mechanisms for growth inhibition, epithelial-mesenchymal transitions (EMTs) and migration of the hepatoma cell line HepG2 triggered by the phorbol ester tumor promoter TPA (12-O-tetradecanoyl- phorbol 13-acetate). The transcriptional factor, Snail, has been previously shown to be responsible for reduction of gene expression of the cell-cell adhesion molecule E-cadherin resulting in EMT and cell migration. On the other hand Snail may also trigger cell cycle arrest by down-regulation of Cyclin D1.
In this study, we found Snail can be induced by TPA and antisense Snail oligodeoxynucleotides can simultaneously prevent all the cellular effects of TPA on HepG2 including growth inhibition, EMT and migration of HepG2. Consistently, the preventive effect of AS snail ODNs on TPA-induced down regulation of cell-cell adhesion molecule E-cadherin was observed. On the other hand, in HepG2cell overexpressing Snail we found TPA-induced upregulation of the CDK inhibitor p15INK4b and downregulation of Cyclin D1 were also attenuated by transfection of AS Snail.
We also established HepG2 clone stably overexpressing Snail for investigating whether Snail alone is sufficient for regulation of migration and growth of HepG2. We observed that Snail-overexpressing HepG2 acquired higher migratory ability than parental HepG2, however, E-cadherin expression was not decreased. On the other hand, Snail-overexpressing HepG2 exhibit higher doubling time of cell growth and increased G1 phase of the cell cycle accompanied with enhanced p15INK4b gene expression, however, decreased Cyclin D1 was not observed.
In conclusion, the induction of Snail was required not only for TPA-suppressed gene expression of E-cahedrin resulting in EMT and migration but also for upregulation of CDK inhibitor and downregulation of Cyclin D1 leading to G1 cell cycle arrest of HepG2 triggered by TPA. On the other hand, Snail seems to be sufficient for triggering growth retardation and cell migration of HepG2. On the gene level, Snail was sufficient for induction of p15INK4b but not for suppression of E-cahedrin and Cyclin D1. In the future more experiments should be done to investigate the role of additional genes such as Fibronectin, Integrin , MMPs responsible for snail-triggered EMT and migration in HepG2.
英文摘要…………………………………………………………………………….. 1
中文摘要…………………………………………………………………………….. 3
壹、緒論………………………………………………………………………….. 5
貳、實驗材料與方法…………………………………………………………….. 10
參、實驗結果…………………………………………………………………….. 27
肆、結論與討論………………………………………………………………….. 36
伍、參考文獻…………………………………………………………………….. 41
陸、圖表目錄
圖一………………………………………………………………………….. 47
圖二………………………………………………………………………….. 48
圖三、圖四………………………………………………………………….. 49
圖五………………………………………………………………………….. 50
圖六………………………………………………………………………….. 51
圖七………………………………………………………………………….. 52
圖八………………………………………………………………………….. 53
圖九………………………………………………………………………….. 54
圖十………………………………………………………………………….. 55
圖十一……………………………………………………………………….. 56
圖十二……………………………………………………………………….. 57
圖十三……………………………………………………………………….. 58
表一………………………………………………………………………….. 59
圖十四……………………………………………………………………….. 60
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