肝癌是一種高發生率及高死亡率的癌症，死亡率在全世界癌症死因中排名第三，此外，肝癌在國人癌症死因中占男性死因第一位，女性死因第二位，因此肝癌的治療是迫切需要研究的議題。Cisplatin (順鉑) 是一種廣效型的抗癌藥物，但使用上有劑量限制性，高劑量使用cisplatin會產生腎毒性副作用。近年來有許多文獻指出，藉由降低cisplatin劑量之使用並與天然物或營養素合併，能達到加乘抗癌之效果。香杉芝為薄孔菌屬，於2004年被鑑定為新種，是台灣特有之真菌。有學者發現香杉芝萃取全液具抗氧化、抗發炎及抗癌功效，且香杉芝全液在活體內外皆有抗癌作用。然而關於香杉芝的文獻極少，確切機制尚待研究。本實驗利用香杉芝菌絲體乙醇萃取物 (AsE) 單獨或合併抗癌藥物cisplatin，對具高度轉移性之人類肝癌細胞株SK-Hep-1作用，探討單獨及合併之凋亡機制與加乘效應。
　　MTT實驗結果顯示，隨著濃度及作用時間增加，AsE顯著抑制SK-Hep-1之細胞增生，且25、75及125 μg/mL之AsE合併cisplatin (40 μM) 於48小時培養條件下，抗增生作用具相加效應，接著探討肝癌細胞凋亡之機制。實驗結果發現AsE能誘導SK-Hep-1之DNA受損，而誘使細胞死亡主要機制包括使細胞之sub-G1期增加、細胞凋亡數增加並活化癌細胞之caspase，進而產生抗癌作用。進一步將cisplatin合併AsE共同培養，結果發現，細胞凋亡比率顯著上升，且皆具相乘效應 (Fold of synergy分別為1.78、1.84及1.09)；cisplatin合併125 μg/mL AsE培養48小時後，sub-G1期顯著增加；cisplatin合併75及125 μg/mL之AsE並與SK-Hep-1共同培養48小時後，細胞拖尾顯著增加，DNA受損加劇，尾矩之增加具相乘效應 (Fold of synergy分別為1.12及1.31)；此外，caspase表現量亦顯著增加，合併125 μg/mL AsE培養48小時，caspase-3活性有相乘效應 (Fold of synergy = 1.27)。
　　綜合上述結果，本研究證實香杉芝菌絲體乙醇萃取物可有效地抑制人類肝癌細胞株SK-Hep-1之增生，其作用機制主要是藉由誘發癌細胞產生凋亡，且cisplatin與AsE合併會使SK-Hep-1停滯在G0/G1期、出現sub-G1期延長、造成DNA之損傷並誘使caspase活化，最終使癌細胞走向凋亡，達到抗癌目的。並由caspase活性分析結果推測，單獨AsE培養，凋亡可經由內源性或外源性路徑產生，合併cisplatin後，內源性凋亡路徑機制增加。因此，以香杉芝菌絲體乙醇萃取物為佐劑合併cisplatin在促使肝癌細胞產生凋亡上具有潛力，期望可作為天然物抗肝癌上之新選擇。

Hepatocellular carcinoma (HCC) is the high incidence and high mortality cancer which is the third cause of death from cancer in the world. HCC is also the first cause of cancer death in men and the second cause of cancer death in women in Taiwan; therefore, the novel treatment of HCC is an imperative need to develop. Cisplatin is one of the most widely used anti-cancer agents, but high dose administered to patient produce nephrotoxicity side effect and the dose of cisplatin must be limiting. Recently, studies have suggested that nature compounds or nutrients combined with low-dose cisplatin result a synergistic anti-cancer activity. Antrodia salmonea is a new species of the Antrodia and was first identified in 2004 which is only collected from the Cunninghamia konishii Hayata in Taiwan. Studies found that the extract of A. salmonea exhibited the anti-oxidative, anti-inflammation, anti-cancer ability and cytotoxic activity in vitro and in vivo. However, there is little information reported in the literature and the mechanisms underlying are still not clear. In this study, we investigated the apoptotic effect of the ethanol extract from Antrodia salmonea mycelia (AsE) in SK-Hep-1 cells, a highly invasive human hepatoma cell lines, and the effect of the combination of AsE and cisplatin and the possible mechanisms.
　　The results showed that the cell viability of SK-Hep-1 was concentration- and time-dependent suppression. At the 25, 75 and 125 μg/mL concentration of AsE combined with cisplatin in 48 h incubation had additive anti proliferation effect. We found that AsE induced apoptosis in SK-Hep-1. The main mechanism of the action included the cell cycle arrested at G0/ G1 phase, DNA damage and the activation of caspases. Furthermore, we combined AsE with cisplatin (40 μM) and found the synergistic increase in the rate of apoptosis. In 48 h incubation, a significant raise in sub-G1 phase of the combination of 125 μg/mL AsE and cisplatin. After treatment with cisplatin in combination with 75 and 125 μg/mL AsE for 48 h, significant DNA damage appeared and there was the synergistic damage to DNA. Otherwise, the 125 μg/mL concentration of AsE in combination with cisplatin increased caspases activity significantly, especially in 48 h, which synergistically promoted caspase-3 activity.
　　In conclution, we demonstrated that AsE inhibit the cell proliferation effectively in Sk-Hep-1 cells by inducing apoptosis. AsE in combination with cisplatin blocked the cell cycle progression at the G0/ G1 phase, elevated sub-G1 phase, induced DNA damage, activated caspases and culminated in the activation of apoptosis. AsE triggers apoptosis through intrinsic and extrinsic pathways, and the intrinsic pathway increase after combined with cisplatin. The combination of AsE and cisplatin exhibited a enhanced cytotoxic effect in SK-Hep-1 cells. These results warrant further studies on AsE as a potential adjuvant chemotherapeutic agents.

表次 vii
圖次 ix
縮寫表 xii
前言 1
文獻整理 3
一、肝癌 3
二、細胞凋亡 4
(一)凋亡指標 4
(二)Caspase-3 7
(三)Caspase-8 8
(四)Caspase-9 8
三、細胞週期 9
(一)CDKs 9
(二)Cyclins 10
(三)CKI 11
四、香杉芝 12
(一)簡介 12
(二)成分結構 12
(三)功能 14
五、抗癌藥物 16
(一)Cisplatin 16
(二)Doxorubicin hydrochloride 18
(三Oxaliplatin 19
研究動機 21
實驗架構 22
材料與方法 23
一、實驗材料 23
(一)菌絲體樣品粉末 23
(二)細胞株 23
(三)試藥 23
二、實驗方法 24
(一)樣品製備 24
(二)細胞株培養 25
(三)細胞存活率測定 26
(四)彗星試驗 26
(五)細胞週期 27
(六)細胞凋亡 27
(七)Caspase活性分析 28
(八)加乘效應 29
(九)統計分析 29
結果與討論 30
一、細胞存活率試驗 30
二、彗星試驗 49
三、細胞週期分析 57
四、細胞凋亡試驗 68
五、Caspase-3, -8, -9活性分析 74
結論 90
參考文獻 91

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