臺灣博碩士論文加值系統

癌症是現今許多國家中重要的問題之一，近年來癌症是台灣十大死亡原因的首位。然，肺癌是全部癌症死亡人口中的第一位，每年全世界約將近一百萬人死於肺癌。目前針對肺癌臨床的治療策略被相繼的發展出來，而發展出針對治療固態性癌症的機轉除了促使癌細胞產生凋亡反應外，另一機轉則是抑制癌細胞的血管增生；或者同時並有促使癌細胞產生凋亡與抑制癌細胞的血管增生。
於我們的研究中，以非小細胞型肺癌（Non-small cell lung cancer），A549 細胞株當做標的細胞，將多種合成化合物做初步抗癌活性篩選，用MTT分析其細胞毒殺率。由實驗結果發現，GO-13是最具有癌細胞毒殺作用的化合物。由lactate dehydrogenase釋放作用實驗、 Annexin-V-labeling和TUNEL的強螢光免疫反應及顯微鏡下的細胞型態觀察，得知GO-13合成化合物可促使非小細胞型肺癌細胞產生細胞凋亡。其半致死量（IC50）是 6.8 μM，而細胞凋亡作用具有時間與濃度依賴性（time and concentration—dependent）。更進一步以功能性的定量研究及西方墨點法分析發現，經GO-13合成化合物誘導非小細胞型肺癌細胞所產生細胞凋亡的機制中除了自由基有參與調控外，Ras、 p38 mitogen-activated protein kinase、protein kinase C 和phospatidylinositol 3-inase並沒有扮演重要的調控角色。
由西方墨點法實驗中得知：非小細胞型肺癌細胞本身並沒有BcL-2
蛋白的表現。然而，我們可明顯的觀察到經由GO-13所誘導產生細胞凋亡的機制中，短時間的作用（3小時內）便可使Bcl-XL蛋白的表現減少，而長時間（大於7小時）可促使Bax蛋白表現增加；此外，GO-13也會活化caspase-3 的活性。有趣的是，我們於血管增生的裸鼠實驗中觀察到，GO-13可有效抑制vascular endothelium growth factor引發的血管增生作用。
總結：GO-13可經由引發Bcl-2 down-regulation、Bax up- regulation及caspase-3 的活化，而來引起A549細胞的凋亡作用。此外，GO-13在裸鼠的動物模式，也能有效的抑制血管新生作用。

Cancer is one of the serious problems in many countries. In the recent years, it has been the most important causative factor causing deaths in Taiwan. Lung cancer is one of the predominant leading causes in human cancer death, accounting for the lives of more than a million people yearly worldwide. Therefore, several strategies for the clinical therapies have been raised. For the treatment of solid tumors, the chemotherapies, such as the apoptotic and antiangiogenetic agents or the combination of both are developed. In the present work, we examined the synthetic compounds obtained form Dr. Chern on the cytotoxic activities in human lung carcinoma A549 cells by MTT assay. The data demonstrated that GO-13 is the most effective one among these compound to induce the cytotoxic effect in A549 cells. Furthermore, GO-13 induced cytotoxicity was identified to be the apoptotic reaction base on several criteria: negative release reaction of lactate dehydrogenase (LDH), morphology characterization, positive Annexine-V-labeling and TUNEL response. GO-13 induced the apopototic reaction in a time and concentration dependent manner with an IC50 value of 6.8 μM.
Furthermore, using several functional assays and Western blotting detection, the results demonstrated that the regulation of Ras, p38 mitogen-activated protein kinase, protein kinase C and phosphatidylinosi-
tol 3-kinase activities did not involve in the GO-13-mediated apoptotic mechanism but except for the free radical regulation.
In this study, there is no Bcl-2 protein expression and two survival-
related proteins were detected following the administration of GO-13 in A549 cells. Bcl-XL protein is well identified to be the crucial survival protein whereas Bax, the apoptotic protein in a lot of cells. GO-13 induced the significant down-regulation of BcL-XL expression within the short time period (3 hr) and the profound up-regulation of Bax expression in a long-term treatment (more than 7 hr). GO-13-induced apopotosis was also positively correlated with the increase in caspase-3 activity. Interestingly, GO-13 (10 μM) showed a modest but significant inhibition on vascular endothelium growth factor (VEGF)-induced angiogenic effect in nude mice models. In summary, we suggest that GO-13 induced the apoptotic reaction via the BcL-XL down-regulation, Bax up-regulation, and the following caspase-3 activation in A549 cells. Furthermore, GO-13 also exhibits the antiangiogenic effect in the nude mice model.

目錄
縮寫表……………………………………………………………………I
中文摘要………………………………………………………………..IV
英文摘要………………………………………………………………..VI
研究動機與目的……………………………………………………...VIII
文獻回顧…………………………………………………………………1
實驗材料與方法………………………………………………………..22
一、 實驗材料…………………………………………………22
二、 細胞培養…………………………………………………23
三、 實驗方法…………………………………………………28
結果……………………………………………………………………..36
討論……………………………………………………………………..43
結論……………………………………………………………………..51
圖表……………………………………………………………………..52
參考文獻………………………………………………………………..67

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