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研究生:曾耀廣
研究生(外文):Yao Kuang Tseng
論文名稱:DHEA抑制腫瘤細胞生長以及侵犯能力之機轉研究
論文名稱(外文):DHEA-Induced Inhibition of Growth and Invasiveness in Hepatoma Cells: A mechanistic Study
指導教授:何鴻耀
指導教授(外文):H. Y. HO
學位類別:碩士
校院名稱:長庚大學
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
論文出版年:2008
畢業學年度:96
論文頁數:96
中文關鍵詞:粒線體去氧表雄固酮
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去氧表雄固酮 (Dehydroepiandrosterone, DHEA)是體內含量豐富的一種腎上腺荷爾蒙 (adrenal steroid hormone)。過去的研究指出DHEA抑制腫瘤細胞的生長其詳細機制仍不清楚且DHEA是葡萄糖六磷酸去氫酶(G6PD)非競爭型抑制物。實驗室先前的研究已指出去氧表雄固酮 (DHEA)並沒有意志葡萄糖六磷酸去氫酶 (G6PD)的活性。本實驗結果為探討去氧表雄固酮 (DHEA)抑制腫瘤細胞生長。我們發現去氧表雄固酮抑制腫瘤細胞生長 (cell proliferation)和不接觸依賴的生長(anchorage-independent cell growth)。同樣地在DHEA作用下,細胞的移動能力(migration)以及細胞之侵犯能力 (invasiveness)也被抑制。這些結果都顯示去氧表雄固酮 (DHEA)並非抑制葡萄糖六磷酸 (G6PD)去氫酶活性。我們發現去氧表雄固酮 (DHEA)降低細胞粒線體膜電位以及粒線體呼吸率,粒線體功能異常亦造成細胞內的氧化壓力增加。從粒線體基因表現觀察,DHEA可降低如SDHA之表現。綜合上述結果,DHEA抑制腫瘤細胞生長以及轉移能力,可能經由改變粒線體功能以及粒線體之基因表現。
Key word: Dehydroepiandrosterone、glucose-6-phosphate dehydrogenase、Mitochondrion
Dehydroepiandrosterone (DHEA) is an abundant adrenal steroid hormone in human body. Also, Dehydroepiandrosterone (DHEA) is recognized a noncompetitive inhibitor of glucose-6-phosphate dehydrogenase (G6PD). Dehydroepiandrosterone (DHEA) is known to suppress tumor cell proliferation. However, the mechanism is not entirely clear. Our previous study showed that DHEA is not associated with inhibition of glucose-6-phosphate dehydrogenase (G6PD), the putative DHEA target. In the present study, we investigated the mechanism underlying DHEA-mediated growth inhibition. We found that DHEA suppressed proliferation, spheroid formation in semisolid media, migration, as well as invasiveness of G6PD knock-down (SK-GI) and scramble control (SK-SC) cells. These results suggest that DHEA acts independent of G6PD. Interestingly, G6PD knockdown alone leads to reduced proliferation as well as growth in semisolid media. We also demonstrate that DHEA reduced mitochondrial membrane potential and mitochondrial oxygen consumption. We found that DHEA increased reactive oxygen species and mitochondrial mass. Also, we found that DHEA reduced mitochondrial gene expression such as SDHA. Mechanistically, we found that DHEA-mediated effect was associated with mitochondrial anomalies.
Key word: Dehydroepiandrosterone、glucose-6-phosphate dehydrogenase、Mitochondrion
目 錄
指導教授推薦書 ……………………………………………………
口試委員會審定書 …………………………………………………
授權書 ……………………………………………………………… iii
誌謝 ………………………………………………………………… v
中文摘要 …………………………………………………………… vi
英文摘要 …………………………………………………………… vii
目錄 ……………………………………………………………… viii
圖目錄 ……………………………………………………………… xii
第一章 序論…………………………………………………………… 1
1.1 前言…………………………………………………………… 1
1.1.1去氧表雄固酮 (DHEA)………………………………… 1
1.1.2癌症與粒線體 ………………………………………… 3
第二章 材料與方法…………………………………………………… 8
2. 1實驗材料……………………………………………………… 8
2.1.1細胞株…………………………………………………… 8
2.1.2藥品、緩衝液 、培養液、試劑………………………… 8
2.1.2.1細胞培養與DHEA處理……………………………… 8
2.1.2.2 細胞計數………………………………………… 9
2.1.2.3 蛋白質萃取及定量………………………………… 9
2.1.2.4 流式細胞儀、雷射共軛交顯微鏡…………………… 9
2.1.2.5 中性紅 ……………………………………………… 9
2.1.2.6 軟洋菜交測試………………………………………… 11
2.1.2.7 體外劃痕測試………………………………………… 11
2.1.2.8 群落生成測試……………………………………… 11
2.1.2.9 移動能力測試……………………………………… 12
2.1.2.10 侵犯能力測試………………………………………12
2.1.2.11 粒線體呼吸率溶液………………………………… 12
2.1.2.12 細胞能量代謝測定………………………………… 13
2.1.2.13 粒線體活性………………………………………… 13
2.1.2.14 SHE溶液…………………………………………… 14
2.2實驗方法 …………………………………………………… 15
2.2.1細胞培養 ………………………………………………… 15
2.2.2腫瘤細胞之群落生成…………………………………… 15
2.2.3軟洋菜膠測試 ……………………………………………16
2.2.4腫瘤細胞之體外劃痕測試……………………………… 16
2.2.5腫瘤細胞之移動能力測試…………………………………17
2.2.6腫瘤細胞之侵犯能力測試…………………………………18
2.2.7呼吸率測定法 ……………………………………………18
2.2.8細胞萃取粒線體 …………………………………………19
2.2.9粒線體耗氧率………………………………………………20
2.2.10粒線體複合體Ⅰ+Ⅲ活性測定…………………………22
2.2.11粒線體複合體Ⅱ+Ⅲ活性測定…………………………23
2.2.12細胞色素C氧化酶活性測定……………………………24
2.2.13粒線體膜電位測定………………………………………25
2.2.14 共軛焦顯微鏡……………………………………………26
2.2.15 細胞活性氧化物測定………………………………… 26
2.2.16 粒線體質量測定……………………………………… 27
2.2.17 細胞能量測定……………………………………………27
2.2.18 以反轉錄聚合酶鏈反應測粒線體複合體基因表現… 28
2.2.19 統計分析…………………………………………………32
第三章 結果 ……………………………………………………… 33
3.1 DHEA對SK-GI和SK-SC腫瘤細胞生長之影響……………33
3.2 DHEA減低SK-SC和SK-GI不依賴接觸生長………………33
3.3 DHEA降低SK-SC、SK-GI腫瘤細胞之侵犯能力…………35
3.4 DHEA降低SK-SC和SK-GI腫瘤細胞之移動能力測試……35
3.5 DHEA降低SK-SC和SK-GI腫瘤細胞之粒線體膜電位……37
3.6 DHEA降低SK-SC與SK-GI腫瘤細胞之呼吸率……………38
3.7 DHEA增加SK-SC與SK-GI腫瘤細胞之活性氧化物………39
3.8 DHEA增加SK-SC與SK-GI腫瘤細胞之粒線體質量………39
3.9 DHEA影響SK-SC與SK-GI腫瘤細胞之能量代謝……… 40
3.10 DHEA影響SK-SC與SK-GI腫瘤細胞之粒線體基因表現40
第四章 討論…………………………………………………………71
參考文獻 ……………………………………………………………78
圖 ……………………………………………………………………42
表 ……………………………………………………………………70

圖目錄
圖一、不同DHEA濃度對SK-SC、SK-GI之腫瘤細胞之生長影響… 42
圖二、DHEA降低SK-SC、SK-GI腫瘤細胞之不依賴接觸生長…… 44
圖三、DHEA降低SK-SC、SK-GI腫瘤細胞之侵犯能力影響……… 48
圖四、DHEA降低SK-SC、SK-GI腫瘤細胞之移動能力影響……… 50
圖五、DHEA降低SK-SC、SK-GI腫瘤細胞內粒線體膜電位之影響…54
圖六、DHEA降低SK-SC、SK-GI腫瘤細胞之呼吸率的影響……… 58
圖七、DHEA增加SK-SC、SK-GI腫瘤細胞之活性氧化物………… 60
圖八、DHEA增加SK-SC、SK-GI腫瘤細胞之粒線體質量………… 62
圖九、DHEA對SK-SC、SK-GI細胞之能量代謝影響……………… 64
圖十、DHEA抑制SK-SC、SK-GI細胞之粒線體基因表現………… 68
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