臺灣博碩士論文加值系統

中國橄欖(Canarium album L.)的果實或其他部位分離出來的純物質已被證實具有許多生理機能效果，例如護肝、抗氧化等，但目前中國橄欖萃取物對癌細胞影響的研究甚少，而本研究室先前的研究指出，中國橄欖水萃物殘渣，甲醇萃取之乙酸乙酯區分層(COE)具有抑制以小鼠大腸癌細胞CT26誘發之腫瘤及大腸癌細胞增生之功效。在腫瘤形成初期，癌細胞快速增生，內層癌細胞距離血管較遠，難以獲得氧氣及葡萄糖，因此本研究擬進一步探討COE於不同葡萄糖環境中抑制大腸癌細胞的功效以及相關機制。我們使用人類大腸癌細胞HCT116作為模式，分別於高濃度(4.5 g/L)及低濃度(1 g/L)葡萄糖的環境下培養，並給予不同劑量的COE，比較其對細胞存活率、癌化能力、爬行能力的影響。我們發現，的COE在高葡萄糖下能夠有效抑制HCT116，但是在低葡萄糖中，抑制效果反而較差，群落生成試驗中也看到低葡萄糖中COE的抑制能力顯著較差，轉移能力則是不受葡萄糖濃度影響。進一步檢測其中可能的分子機制時發現，在低葡萄糖環境中COE透過大幅增加AMPK磷酸化而提昇其活性，因而藉著增加乙酰輔酶A羧化酶(acetyl-CoA carboxylase，ACC)磷酸化而降低其活性，並使得脂肪酸氧化(fatty acid oxidation，FAO)相關基因表現大幅增加，高葡萄糖中則較不顯著，推斷此為COE在不同葡萄糖濃度中造成不同生長效應的主因。而當COE劑量超過400 μg/ml時開始造成粒線體損傷，推測增加的FAO因而無法透過粒線體產生ATP，因此有毒殺HCT116的效應。雖然近年來有越來越多文獻指出植萃物(phytochemicals)具有抑制癌細胞的潛力，本研究結果顯示出某些植化素在使用上當注意其對不同狀態的癌細胞可能有相反的效應，必須謹慎應用。

The pure compounds isolated from the fruit or other parts of Chinese olive (Canarium album L.) have been proven to have many physiological functions such as liver protection, and anti-oxidation. However, little is known about the effects of Chinese olive extracts on cancer cells. Previous research in our lab indicates that the ethyl acetate fraction of ethanol extract of Chinese olive water extract residue (COE) exhibits anti-tumor effect in both cell and animal models. In the early stage of tumor formation, cancer cells rapidly proliferate, thus makes the inner cancer cells lack the supply of oxygen and glucose. Therefore, this study intends to investigate the anti-tumor effect of COE in either high or low glucose. We have treated HCT116 cells with different concentration of COE at different glucose concentrations, and examined the efficacy of COE on cell viability, colony formation, and migration. We found that COE at effectively inhibit HCT116 cell growth in high glucose. However, in low glucose, the inhibitory effect is rather poor. Also, in clonogenic assay, less inhibitory effect of COE in low glucose was observed. Furthermore, in low glucose, COE could further activate AMPK and the consequent inactivation of ACC as well as the enhancement of fatty acid oxidation (FAO) related genes. This phenomenon was less significant in high glucose, which caused opposite growth effects in different glucose levels upon COE treatment. COE dose exceeds 400 μg/ml would damage mitochondrial and leads to apoptosis, which can no longer rescue cells by the additional function of COE on increasing FAO mediated energy production. Although more and more studies point out that phytochemicals have the potential to inhibit cancer cells, here the results suggested that the opposite effects upon different concentration of glucose should be considered before using phytochemicals for cancer therapy.

謝誌 I
摘要 II
ABSTRACT III
目錄 V
圖目錄 VIII
表目錄 IX
縮寫表 X
第一章 前言 1
第二章 文獻回顧 2
第一節 癌症及其代謝 2
1. 癌化(carcinogenesis) 2
2. 有氧糖解(Warburg Effect) 2
3. 戊糖磷酸化路徑(pentose phosphate pathway) 3
4. AMPK(AMP-activated protein kinase) 路徑 4
5. 脂肪酸代謝(Fatty acid oxidation，FAO) 4
第二節 大腸直腸癌 7
1. 大腸直腸癌分級 7
2. 葡萄糖濃度對大腸直腸癌的影響 8
第三節 粒線體 9
1. 癌細胞的粒線體 9
2. 粒線體失能(mitochondrial dysfunction) 10
第四節 中國橄欖 12
1. 中國橄欖中的成分 12
2. 中國橄欖的生物活性 12
3. 其他橄欖屬(Canarium)相關研究 13
第三章 研究目標與實驗架構 15
第一節 研究目標 15
第二節 實驗架構 16
第四章 材料與方法 17
第一節 實驗材料 17
第二節 實驗方法 21
1. 細胞培養 21
2. 細胞存活率試驗 21
3. 細胞生長曲線 22
4. 細胞凋亡檢測 22
5. BCA蛋白定量法 22
6. 粒線體膜電位檢測 23
7. 細胞總RNA萃取 24
8. DNA/RNA電泳 24
9. 反轉錄 25
10. 即時定量聚合酶連鎖反應 25
11. 群落生成(克隆試驗) 26
12. 傷口癒合試驗 26
13. 西方墨點法 26
14. 統計方法 29
第五章 實驗結果 30
第一節 不同葡萄糖濃度下COE對HCT116生長情形及惡性程度的影響 30
第二節 不同葡萄糖濃度中COE促進脂肪酸氧化的程度不同 39
第三節 COE在不同葡萄糖濃度環境中造成不同程度的細胞凋亡 45
第四節 不同劑量的COE對粒線體造成不同影響，與葡萄糖濃度無關 52
第六章 討論 57
第七章 結論 60
參考文獻 61

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