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研究生:楊嘉銘
研究生(外文):Jia-Ming Yang
論文名稱:中國橄欖萃取物於口腔癌細胞之抗腫瘤機制研究
論文名稱(外文):The study of molecular mechanism underlying the anti-tumor effect of Chinese olive extracts in oral cancer cells
指導教授:林如華
指導教授(外文):Ju-Hwa Lin
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:生物科技學系碩士班
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:2015
畢業學年度:103
語文別:中文
論文頁數:67
中文關鍵詞:口腔鱗狀上皮細胞癌中國橄欖細胞凋亡細胞遷移
外文關鍵詞:Oral Squamous Cell CarcinomaChinese oliveApoptosisCell migration
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口腔鱗狀細胞癌 (OSCC) 是世界上10個最常見的癌症之一,每年都有超過50萬名病患被確診。在台灣口腔鱗狀細胞癌致死率排名約第六,且每年伴隨著5400個新的病例及2200個死亡案例。本實驗採用生長於台灣的中國橄欖 (Canarium album L.) 果實,進行口腔癌抗腫瘤之研究。根據本草綱目記載,中國橄欖果實具有抗菌、抗病毒、抗發炎與解毒之功效。在先前的研究顯示其含有豐富的多酚類物質 (phenolic compounds) 與體外抗氧化的功效成分。然而,目前尚未有研究探討中國橄欖抗癌的功效。在本論文中,我們利用中國橄欖萃取物 (MeOH-EA and MeOH-Bu),研究口腔鱗狀細胞Ca922的抗癌活性及其分子機制。在 MTT assay 中,發現中國橄欖萃取物隨著濃度及時間增加,有抑制Ca922細胞生長的情形,然後再利用細胞凋亡檢測 (Annexin V and PI staining),發現顯著apoptosis情形,最後再利用 western blotting 觀察 apoptosis 相關蛋白的變化量,發現 caspase-9、3、PARP 皆被活化,且 p53蛋白表現量上升。從 wound healing 和 transwell 的實驗中,觀察到中國橄欖萃取物有顯著抑制 Ca922 細胞migration的情形。本論文研究結果顯示,中國橄欖萃取物在口腔鱗狀細胞癌中具有抗腫瘤之作用,並初步了解其作用機轉。

Oral squamous cell carcinoma (OSCC) is one of the 10 most frequent cancers worldwide with more than half a million patients being diagnosed each year. In Taiwan, OSCC has been the sixth leading cause of death from cancer with nearly 5400 new cases and 2200 deaths per year. Chinese olive (Canarium album L.) belongs to Burseraceae family which differs from Mediterranean diet’s olive in type. It is also a traditional medicine material, exhibiting beneficial functions in anti-virus, anti-inflammation and detoxification. Previous studies have displayed Chinese olive a phenolic compounds rich resource for providing antioxidative activity. However, the role of Chinese olive in anti-cancer remains unknown. In this study, we investigated the anti-cancer activity of Chinese olive extracts (MeOH-EA and MeOH-Bu) and the molecular mechanisms in the oral squamous cell carcinoma cell line Ca922. Chinese olive extracts (MeOH-EA and MeOH-Bu) inhibited Ca922 cell viability in a dose and time dependent manner by MTT assay. After treatment with 0.2 mg/ml Chinese olive extracts (MeOH-EA and MeOH-Bu) for 48 h, apoptosis was obviously observed as assayed by Annexin V-FITC/PI staining. Western blotting analysis showed that caspase-9、3 and the cleavage of PARP were activated and p53 expression was up-regulated following 0.2 mg/ml Chinese olive extracts (MeOH-EA and MeOH-Bu) treatment for 24 and 48 h. To evaluate the effects of OSCC on cell migration under treatment with Chinese olive extracts (MeOH-EA and MeOH-Bu), wound healing and a transwell migration assay were performed. The results showed Chinese olive extracts (MeOH-EA and MeOH-Bu) significantly inhibits Ca922 cell migration in a dose-dependent manner. Migration-related proteins were analyzed by western blotting. Taken together, our data indicate that Chinese olive extracts may be a useful effective anticancer agent in the treatment of OSCC.

總目錄
總目錄 I
圖目錄 V
附錄 VII
中文摘要 VIII
Abstract IX
縮寫表 X
第一章 緒論 1
第一節 口腔鱗狀上皮細胞癌 (Oral Squamous Cell Carcinoma) 之介紹 1
第二節 中國橄欖 (Chinese olive) 之介紹 2
第三節 中國橄欖中的成分 3
第四節 中國橄欖的多酚類物質之功效 3
第五節 中國橄欖以不同溶劑萃取及區分層製備 5
第六節 細胞凋亡之機制 6
第七節 細胞週期 (Cell cycle) 之機制 7
第八節 腫瘤抑制蛋白 p53 8
第九節 癌細胞之轉移 9
第二章 研究動機與目的 11
第一節 研究動機 11
第二節 研究目的 11
第三章 結果與討論 12
研究結果 12
第一節 檢測Chinese olive extracts (MeOH-EA and MeOH-Bu) 是否會對人皮膚成纖維細胞 (human skin fibroblast , HSF) 產生毒性 12
第二節 中國橄欖之 methanol 萃取不同區分層的萃取物對於口腔癌細胞株生長之影響 12
第三節 利用不同濃度的Chinese olive extracts (MeOH-EA and MeOH-Bu) 檢測Ca922的細胞存活率 13
第四節 利用Chinese olive extracts (MeOH-EA and MeOH-Bu) 檢測不同時間點對於Ca922存活率之影響 13
第五節 Chinese olive extracts (MeOH-EA and MeOH-Bu) 對Ca922生長型態之影響 14
第六節 以流式細胞儀檢測Chinese olive extracts (MeOH-EA and MeOH-Bu) 對於Ca922引發之細胞凋亡 14
第七節 Chinese olive extracts (MeOH-EA and MeOH-Bu) 干擾Ca922之細胞週期分布 15
第八節 Chinese olive extracts (MeOH-EA and MeOH-Bu) 誘導pro-caspase-9 和 pro-caspase-3裂解 15
第九節 Chinese olive extracts (MeOH-EA and MeOH-Bu) 會增加Caspase-3的活性 16
第十節 Chinese olive extracts (MeOH-EA and MeOH-Bu) 誘導PARP裂解 16
第十一節 Chinese olive extracts (MeOH-EA and MeOH-Bu)對p53、P-p53、Bcl-2和Bc l-xL蛋白表現的影響 17
第十二節 利用wound healing assay來看Chinese olive extracts (MeOH-EA and MeOH-Bu) 對口腔癌細胞Ca922之細胞遷移能力影響 18
第十三節 利用transwell assay來看Chinese olive extracts (MeOH-EA and MeOH-Bu) 對口腔癌細胞Ca922之細胞遷移能力影響 18
第十四節 Chinese olive extracts (MeOH-EA and MeOH-Bu) 誘導E-cadherin的蛋白表現量增加 19
研究討論 20
第四章 結論 22
第五章 實驗部分 23
第一節 研究設計 23
第二節 實驗材料 24
第三節 實驗方法 29
第四節 統計方法 38
第六章 參考文獻 39


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