臺灣博碩士論文加值系統

鹿茸萃取物(Antler’s extract)、靈芝(Ganoderma lucidum)及牛樟芝(Antrodia Camphorata)皆被證實具有多種生物活性因子，包含三萜類、多醣等，這些因子具有抗發炎、抗氧化、抗動脈粥樣硬化及抑制病毒活性等多種療效。然而目前尚未有研究證實將這三種中草藥混合對於口腔癌的治療效果，因此我們將這三種中草藥合成一種名為AGA的強效複合物，並在生物體外和體內研究其對抑制口腔癌的潛力。細胞存活率分析顯示AGA在72小時的口腔癌細胞抑制IC50約為15 mg，因此後續實驗選擇範圍內的10mg及20mg劑量來研究AGA對抑制口腔癌的作用。AGA以劑量和時間依賴性方式顯著抑制口腔癌細胞(HSC3、SAS及OECM-1)的增殖。進一步實驗得知AGA可抑制細胞週期調節因子(CDK4、CDK6、cyclin A、cyclin B1、cyclin D1及cyclin E2)和凋亡抑制蛋白(Bcl-2)，並且能增強促凋亡蛋白(Bax)提升以及促使癌細胞停滯於Sub-G1期。另一方面，AGA抑制了腫瘤轉移相關的EMT標記(N-cadherin、β-catenin及vimentin)，證實它具有降低口腔癌細胞轉移的能力。於動物實驗方面，AGA顯著降低了免疫缺陷鼠(NOD SCID)異種移植口腔癌腫瘤的生長，且沒有肝和腎毒性的不良反應。結論是，我們合成的似雞尾酒療法AGA以三萜類化合物及多醣通過誘導細胞凋亡、抑制遷移及促進Sub-G1期細胞週期停滯進而顯著抑制口腔癌細胞。

Traditional Chinese medicines Antler’s extract (A) and Ganoderma lucidum (G) and Antrodia Camphorata (A) have been known to individually contain a plethora of bioactive factors including triterpenoids, polysaccharides etc., exerting various curative impacts such as anti-inflammatory, anti-oxidative, anti-atherosclerotic and anti-viral activities. However, their combinatorial therapeutic efficacy for oral cancer has not been investigated. Hence, we synthesized a robust cocktail called AGA and investigated its anti-oral cancer potential in vitro and in vivo. An MTT assay revealed the IC50 of AGA to be about 15 mg at 72 h. Therefore, 10 mg and 20 mg doses were selected to study the effect of AGA. The AGA significantly inhibited proliferation of oral cancer cells (HSC3, SAS, and OECM-1) in a dose- and time-dependent manner. AGA retarded cell cycle regulators (CDK4, CDK6, cyclin A, B1, D1 and E2) and apoptosis inhibitory protein Bcl-2, but enhanced pro-apoptotic protein Bax and a higher percentage of cells in Sub-G1 phase. Mechanistically, AGA suppressed all EMT markers; consequently, it decreased the migration ability of cancer cells. AGA significantly reduced xenograft tumor growth in nude mice with no adverse events in liver and renal toxicity. Conclusively, AGA strongly inhibited oral cancer through inducing apoptosis and inhibiting the migration and promotion of cell cycle arrest at subG1 phase, which may be mediated primarily via cocktail-contained triterpenoids and polysaccharides.

致謝 i
中文摘要 ii
目錄 iii-v
圖表目錄 vi
1. 簡介
1.1 癌症簡介 1
1.2 口腔癌
1.2.1 口腔癌流病學 1
1.2.2 口腔癌致病因素 2
1.2.3 口腔癌診斷 2
1.2.4 口腔癌治療 2
1.3 AGA複合物
1.3.1 鹿茸萃取物(Antler’s extract) 2
1.3.2 靈芝(Ganoderma lucidum) 3
1.3.3 牛樟芝(Antrodia Camphorata) 3
1.4 實驗目的 3
2. 材料方法
實驗材料
2.1 實驗細胞株 4
2.2 細胞培養、繼代、凍存相關試劑 4
2.3 AGA來源 4
2.4 細胞存活率試劑 4
2.5 流式細胞儀試劑 5
2.6 西方墨點法試劑 5
2.7 抗體 5
2.8 聚合酶連鎖反應試劑及引子 5
2.9 實驗動物來源與取得 6
實驗方法
2.10 細胞培養與凍存 7
2.10.1 細胞繼代培養 6-7
2.10.2 細胞計數 7
2.10.3 細胞凍存 7
2.11 細胞存活率分析 MTT assay 7
2.12 細胞群落實驗 Colony formation assay 8
2.13 細胞傷痕癒合實驗 Wound healing assay 8
2.14 細胞遷移實驗 Transwell assay 8
2.15 流式細胞儀 Flow cytometry 9
2.15.1 細胞週期分析 Cell-cycle assay 9
2.15.2 細胞凋亡分析 Apoptosis assay 9
2.16 聚合酶連鎖反應 Polymerase chain reaction
2.16.1 RNA萃取及濃度測定 9
2.16.2 反轉錄聚合酶連鎖反應 9-10
2.16.3 即時聚合酶連鎖反應 10
2.17 西方墨點法 Western blot
2.17.1 蛋白質濃度製備 10
2.17.2 蛋白質樣品測定 10-11
2.17.3 膠體電泳、跑膠、轉漬、顯影 11
2.18 動物實驗
2.18.1 小鼠口腔癌腫瘤誘導 12
2.18.2 腫瘤組織紀錄 12
2.18.3 肝、腎毒性測試 12
3. 結果
3.1 AGA複合物抑制口腔癌細胞存活率及群落生長能力 13
3.2 AGA複合物影響口腔癌細胞調節細胞週期及凋亡因子表現 13-14
3.3 AGA複合物影響口腔癌細胞之細胞週期滯留及凋亡 14
3.4 AGA複合物抑制口腔癌細胞轉移 14-15
3.5 AGA複合物影響口腔癌細胞之EMT及其調節因子表現 15
3.6 AGA複合物抑制小鼠腫瘤生長 15-16
4. 討論 17-20
5. 結論 21
6. 圖表 22-28
7. 參考文獻 29-32

圖表目錄
Figure 1. 22
Figure 2. 23
Figure 3. 24
Figure 4. 25
Figure 5. 26
Figure 6. 27
Figure 7. 28

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