近年來，許多海洋天然物或其衍生物已被科學家證實具有豐富的生物活性，特別是做為抗癌藥物。肝癌是世界上最常發生的致死癌症之一，在台灣及許多開發中國家與都造成嚴重的負擔。然而，目前的抗肝癌治療卻也存在許多副作用的問題。先前有研究指出台灣產指型軟珊瑚Sinularia flexibilis萃取物具有抗癌功效。因此，本研究以取自同種類珊瑚的萃取物WY-12來分析其抗肝癌與抗血管新生之作用。實驗結果發現，WY-12可以顯著地抑制人類肝癌細胞SK-hep1的存活、移動、侵犯、聚落生成和非貼附型生長的能力，並發現WY-12會促進SK-hep1細胞有絲分裂活化蛋白質激酶酶（mitogen-activated protein kinase, MAPK）之p38 MAPK磷酸化與增加第一型血紅素氧化酶（heme oxygenase-1, HO-1）與上皮細胞鈣粘蛋白（epithelial cadherin, E-cadherin）蛋白質表現量；並可抑制細胞外訊號調節激酶（extracellular signal-regulated kinase, ERK）活化與纖維狀肌動蛋白（fibrous actin, F-actin）的表現。此外，亦發現到WY-12可以顯著地抑制人類臍靜脈內皮細胞EA.hy926的存活與移動，且可抑制eNOS蛋白質表現量。在活體的實驗中也發現，WY-12可以抑制斑馬魚仔魚的體節間血管的血管新生。綜合以上結果顯示，WY-12具有多功能性抗肝癌的功效，以及同時具有抑制血管新生的潛力。

In recent years, many marine natural products and their derivatives were found to have a variety of biological activities, especially for cancer drugs. Hepatocellular carcinoma (HCC) is one of the most common lethal cancers in the world, and become a heavy burden in many developing countries, including Taiwan. However, there are many side effects exist in current therapies of HCC. The extracts form soft coral Sinularia flexibilis were reported to have anti-cancer effects, and WY-12 is one of the compound origins from the soft coral. Hence, the aim of this study is to analyze whether WY-12 has the properties of anti-HCC and anti-angiogenesis. The present study shows that WY-12 could inhibit viability, migration, invasion, colony formation and anchorage-independent growth of the human HCC cell, SK-hep1. Western blotting and immunocytochemistry analysis indicated that WY-12 increased the level of pp38, HO-1 and E-cadherin, but decreased the expression of pERK and F-actin. Besides, WY-12 could also reduce viability and migration of the human umbilical vein endothelial cell line, EA.hy926, and suppress the expression of eNOS protein. The zebrafish data suggests that WY-12 has anti-angiogenesis effect. In conclusion, these results demonstrate that WY-12 seems to have multiple anticancer effects in HCC, and have the potential for anti-angiogenesis.

論文審定書................................................................................................i
誌謝..........................................................................................................ii
摘要..........................................................................................................iii
Abstract....................................................................................................iv
目錄..........................................................................................................v
圖次..........................................................................................................viii
表次..........................................................................................................x
英文縮寫對照表..........................................................................................xi
前言..........................................................................................................1
流行病學統計.........................................................................................1
肝癌之危險因子......................................................................................1
癌症的特徵............................................................................................1
肝癌的訊息傳遞......................................................................................2
肝癌之臨床治療......................................................................................4
海洋藥物與癌症......................................................................................5
研究動機與目的......................................................................................8
實驗材料與方法..........................................................................................9
細胞種類與培養條件...............................................................................9
細胞繼代培養.........................................................................................9
細胞計數...............................................................................................9
細胞存活率測試.....................................................................................10
細胞型態觀察........................................................................................10
免疫細胞化學染色（immunocytochemistry）...........................................10
細胞聚落形成（colony formation assay）................................................11
軟性瓊脂膠體聚落形成試驗（soft agar colony formation assay）...............11
傷口癒合細胞移動能力試驗（wound healing assay）................................12
Boyden chamber細胞侵犯（invasion）能力試驗.......................................12
Boyden chamber細胞移動能力測試.........................................................13
細胞凋亡測試（顯微鏡觀察）.................................................................13
細胞凋亡測試（流式細胞儀）.................................................................14
Caspase 3活性測試...............................................................................14
核內蛋白質萃取（nuclear extraction）....................................................15
西方點墨法（Western blot）..................................................................16
斑馬魚種魚飼養.....................................................................................17
斑馬魚胚胎取得.....................................................................................18
斑馬魚藥物處理.....................................................................................18
觀察斑馬魚體節間血管新生.....................................................................18
數據統計分析........................................................................................18
實驗結果...................................................................................................19
一、WY-12對肝癌的抑制作用...................................................................19
WY-12對人類肝癌細胞SK-hep1存活的影響..........................................19
WY-12對人類肝癌細胞SK-hep1存活相關之蛋白質表現的影響.................20
WY-12對人類肝癌細胞SK-hep1聚落生長之影響....................................21
WY-12對人類肝癌細胞SK-hep1移動與侵犯（invasion）之影響...............22
WY-12對結締組織生長因子促進肝癌細胞SK-hep1移動之抑制作用..........23
WY-12對肝癌細胞SK-hep1之HO-1蛋白質表現的影響.............................24
二、WY-12對血管新生的抑制作用.............................................................24
WY-12對人類臍靜脈內皮細胞株EA.hy926存活之影響............................24
WY-12對人類臍靜脈內皮細胞株EA.hy926移動能力之影響......................25
WY-12對基因轉殖Tg(fli1:EGFP)血管螢光斑馬魚胚胎之體節間血管
(intersegmental vessel, ISV)形成的影響...............................................25
WY-12對肝癌細胞SK-hep1條件培養基（conditioned medium, CM）誘導
人類臍靜脈內皮細胞株EA.hy926移動的影響..........................................26
WY-12對人類臍靜脈內皮細胞株EA.hy926之eNOS蛋白質表現的影響.......26
討論...........................................................................................................59
WY-12對於人類肝癌細胞SK-hep1存活的抑制作用......................................59
WY-12導致人類肝癌細胞SK-hep1凋亡......................................................60
WY-12對於人類肝癌細胞SK-hep1聚落生長的抑制作用...............................63
WY-12對於人類肝癌細胞SK-hep1移動與侵犯的抑制作用............................64
WY-12對結締組織生長因子促進肝癌細胞移動的抑制作用...........................65
HO-1與肝癌之交互作用...........................................................................66
WY-12對血管新生的抑制作用..................................................................67
總結..........................................................................................................71
未來展望....................................................................................................71
參考文獻....................................................................................................72

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中華民國100年死因統計（民國101年12月）【電子書】。台北市：行政院衛生署。民國102年5月取自：http://www.doh.gov.tw/CHT2006/DM/DM2_2.aspx?now_fod_list_no=12743&class_no=440&level_no=4