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研究生:吳唯慶
研究生(外文):Wei-Ching Wu
論文名稱:Reversine做為口腔癌治療藥物之評估
論文名稱(外文):Evaluation of Reversine as an Anticancer Drug in Oral Cancer
指導教授:陳浩仁李英瑞李英瑞引用關係
指導教授(外文):Hau-Ren ChenYing-Ray Lee
口試委員:黃憲斌洪千惠陳逸夫
口試日期:07112011
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:67
中文關鍵詞:口腔癌
外文關鍵詞:reversineoral cancer
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檳榔已被證實會引發口腔癌,而在台灣,口腔癌在男性的好發率約為11%。目前主要治療口腔癌的方式為手術切除或放射治療,通常也會合併化學藥物如5-Fu及Cisplatin進行治療,但是其5年存活率依然小於30%,因此找尋一個適合的口腔癌治療藥物是相當重要的。而Reversine為合成的類嘌呤小分子藥物,原先主要的用途為刺激已分化的細胞轉變為未分化的細胞。在先前的研究指出,reversine為aurora kinases的抑制劑,並且能夠使血癌細胞的生長下降。在此研究中,我們使用兩種口腔鱗狀上皮細胞癌(oral squamous cell carcinoma,OSCC) OC2及OCSL來觀察reversine是否會對細胞產生影響。我們首先觀察reversine對於細胞生長的影響,發現reversine可以有效的降低細胞存活的數量。我們接著觀察細胞的生長週期,發現reversine可以使細胞週期停留在G2/M期,並且使Cyclin B1的表現時間延長。更進一步地發現,reversine促使細胞無法重新表現Cyclin D1,這些結果暗示了細胞週期可能因此無法重新回到G0/G1期而進入下一輪的複製。再進一步以流式細胞儀偵測後發現,reversine會導致細胞產生細胞凋亡,利用西方點墨法深入分析後發現reversine可以誘導細胞進行第二型質外細胞凋亡路徑(type 2 extrinsic caspase-dependent apoptosis)。此外,我們也發現了reversine可以透過抑制Akt/mTOR 訊息傳遞路徑來誘導細胞產生細胞自噬(autophagy),並且也發現了細胞自噬現象主導了細胞的死亡。綜合以上的結果,我們認為reversine或許具潛力可以成為新的口腔癌治療藥物。
Betel quid-chewing has been proved to induce oral cancer. The incidence of oral cancer is about 11% of male population in Taiwan. The effective therapies in oral cancer treatment are surgical excision and radiation. Then, other anticancer agents such as 5-Fu and Cisplatin were also used in oral cancer chemotherapy. However, the five-year survival rate is still less than 30%. Therefore, to identify an effective drug in oral cancer treatment is very important for increasing the survival rate of patients. Reversine is a synthetic purine-like small molecular, which is first identified as a de-differentiation agent in murine myoblast cell lines. Previous study showed that reversine was an aurora kinases inhibitor and prohibited the survival of leukemia cells. In this study, two oral squamous cell carcinoma (OSCC) cell lines, OC2 and OCSL, were used to evaluate the influence of reversine in oral cancer cells. Firstly, we analyzed the cell survival rate by cell counting after reversine treatment, and found that the cell number of OSCC was significantly reduced. Cell cycle analysis and the western blot showed that reversine induced cell cycle to be arrested in G2/M phase and prolonged expression of Cyclin B1. Moreover, Cyclin D1 could not be re-expressed in reversine-treated OSCC cells, suggesting that whole cell cycle was blocked and the cells can’t re-enter the next G0/G1 phase. Furthermore, flow cytometry analysis demonstrated that apoptosis was induced after reversine treatment. Western blot analysis further elucidated that reversine can induce caspase-dependent cell death through activation of the type 2 extrinsic apoptosis pathway. In addition, autophagy was activated by reducing of Akt/mTOR pathway in reversine-treated OSCC. We also found that autophagy reduce cell viability. Taken together, our results suggest that reversine might be a novel anticancer agent in oral cancer therapy.
中文摘要 2
Abstract 3
第一章. 前言 6
1.口腔癌介紹 6
2.口腔癌與細胞週期 7
3. Aurora kinase與細胞週期 9
4. Aurora kinases與癌症 10
5. Reversine 10
6. 細胞凋亡(Apoptosis) 11
7. 細胞自噬 (Autophagy) 12
研究動機與目的 14
第二章. 材料與方法 15
1. 細胞株與藥品 (Cell lines and chemicals) 15
2. 細胞蛋白萃取 (Cell lysate preparation) 15
3. 西方點墨法 (Western blot) 15
4. 細胞存活分析 (Cell viability analysis) 16
5. 細胞週期分析 (Cell cycle analysis) 16
6. Annexin V與PI雙染 (Annexin V and PI dual staining) 17
7. DNA片段化分析 (DNA fragmentation assay) 17
8. 細胞轉染技術 (Cell transfection) 18
9. 共軛焦顯微鏡分析 (Confocal analysis) 18
第三章. 結果 20
1. 評估OSCC細胞內的aurora kinases之表現量 20
2. Reversine對於OSCC細胞生長的影響 20
3. Reversine對於細胞週期的影響 20
4. 觀察reversine所造成的細胞死亡情況 21
5. Reversine會造成細胞進行質外caspase-dependent細胞凋亡 22
6. Reversine會使細胞進行細胞自噬 23
7. Reversine所引發的細胞自噬會導致細胞死亡 24
8. Reversine所引發的細胞自噬是經由Akt/mTOR 路徑 24
第四章. 討論 26
1. Reversine所導致的口腔癌細胞數量下降 26
2. Reversine造成OSCC細胞停留於G2/M期 27
3. Reversine使OSCC細胞發生質外caspase-dependent細胞凋亡以及細胞自噬 29
第五章. 參考資料 32
第六章. 圖與說明 37
圖1. OCSL及OC2兩種OSCC細胞中皆有磷酸化aurora kinase的表現。 37
圖2. Reversine可以降低細胞存活的數量。 38
圖3. Reversine可以造成細胞週期停留在G2/M期。 40
圖4. Reversine可以影響細胞週期調控蛋白之表現。 41
圖5. Reversine可以誘導細胞發生細胞凋亡。 43
圖6. Reversine造成細胞進行caspase-dependent細胞凋亡。 46
圖7. 細胞加入Z-VAD-fmk後可以減緩reversine誘導的細胞死亡。 47
圖8. Reversine可以誘導細胞產生細胞自噬。 51
圖9. Reversine誘導的細胞自噬導致細胞死亡。 54
圖10. Reversine可以抑制Akt/mTOR訊息傳遞路徑。 56
圖11. Reversine對於MAPK家族蛋白沒有抑制效果。 58
第七章. 附錄 59
1. Antibody 59
2. Reagent & Kit 61
3. Buffer & Solution 63

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