在台灣，癌症為十大死因之首，且大腸癌為十大癌症死因之第三名。大腸癌的發病因素和飲食習慣以及生活模式有很大的關聯性，且因罹患大腸癌而死亡的人數也逐年增加，因此開發有效的大腸癌治療藥物是一個重要的議題。牛樟芝為一多孔菌屬之真菌植物，因生長於樟樹的腐木上而得名。先前的研究指出，牛樟芝可應用於藥物中毒、腹瀉、高血壓等的預防及治療上。近來研究發現，牛樟芝對於許多癌細胞有良好的治療效果。因此，了解牛樟芝萃取物是否可有效對抗大腸癌細胞是本研究之目的。利用細胞存活率方式分析，我們發現牛樟芝成分antroquinonol衍生物AC009可以較顯著抑制大腸癌細胞 (DLD-1、RKO、HCT116及SW480) 的生長。同時，AC009會抑制大腸癌細胞之DNA生合成並造成細胞週期G0/G1期阻滯及細胞凋亡。我們進一步利用西方墨點法確認AC009所造成的細胞凋亡是屬於caspase–dependent 路徑。在機制探討上，我們發現AC009會減少磷酸化Erk1/2及JNK1/2/3以及Bcl-2蛋白質表現量，指出AC009造成細胞凋亡可能是透過MAPK路徑。在活體實驗中，異種移植實驗結果顯示AC009具有良好的抗腫瘤生長能力。綜合上述結果顯示，牛樟芝萃取物AC009是一個具有對抗人類大腸癌細胞潛力的藥物。

Cancer is the most common cause of death and colorectal cancer (CRC) is the third leading cause of cancer death in Taiwan. The risk factors of CRC are dietary and lifestyle factors. Unfortunately, the number of deaths due to CRC has increased in recent years. Thus, it is important to develop an effective therapeutic strategy against CRC. Antrodia cinnamomea (AC) is a plant that belongs to polyporaceae, its name comes by that it grows on the rotten woods of the camphor trees. Previous studies have shown that AC can prevent or treat poisoning, diarrhea, and hypertension. Recently, the therapeutic potential of AC has been reported in various types of cancer. Therefore, this study aims to investigate the anti-tumor effects of AC extract on CRC cells. By using cell viability assay, we found that AC009 antroquinonol derivative reduced the viability of several human CRC cell lines, including DLD-1, RKO, HCT116, and SW480. Moreover, AC009 treatment resulted in a decrease in DNA synthesis, G0/G1 phase arrest, and apoptosis. Furthermore, we found that AC009-induced cells apoptosis was caspase-dependent. In addition, AC009 treatment reduced the protein levels of phospho-Erk1/2, phospho-JNK1/2/3, and Bcl-2 showing that AC009-induced apoptosis may be via MAPK pathway. Moreover, we demonstrated that AC009 could significantly inhibit in vivo tumor growth in xenograft mice models. Taken together, our results suggest that AC009 have the therapeutic potential against human CRC cells.

目 錄
目錄......................................................................................................................I
圖目錄...................................................................................................................IV
中英文名詞對照表…………………………………………………..…….……..V
壹、中文摘要.........................................................................................................VI
貳、英文摘要........................................................................................................VII
參、研究背景............................................................................................................1
1、大腸癌（Colorectal cancer）……………………………………………....…1
 大腸癌簡介………………………………………………………………….1
 流行病學…………………………………………………………………….1
 大腸癌之成因……………………………………………………………….1
 大腸癌之症狀……………………………………………………………….3
 大腸癌之治療分期………………………………………………………….3
2、牛樟芝（Antrodia cinnamomea）…………………………………………....4
 牛樟芝背景簡介…………………………………………………………….4
 牛樟芝抗癌研究…………………………………………………………….4
3、細胞凋亡相關機轉………………………………………….………….………5
 Caspase pathway之功能與調控…………………………………………..5
 Bcl-2家族之功能與調控…………………………………………………..5
 MAPK pathway之功能調控………………………………………………….6
肆、研究動機與目的……………………………………………………………..7
伍、實驗材料與方法……………………………………………………………..8
一、材料……………………………………………………………………………8
＊大腸癌細胞株特性比較………………………………………………………9
二、實驗方法……………………………………………………………………..10
1、細胞培養（Cell culture）………………………………………………….10
2、細胞存活率測試（Cell viability assay）………………………………….11
3、半數抑制濃度計算（IC50 calculate）………………………………….….12
4、細胞增生活性測定（Bromodeoxyuridine assay, BrdU）………………... 12
5、細胞週期測定（Cell cycle assay）………………………………………..13
6、細胞凋亡測試（Annexin V FITC apoptosis assay）……………………...15
7、乳酸去氫酶釋放量測試（Lactate dehydrogenase releasing assay, LDH）16
8、西方墨點法（Western blotting）………………………………………….17
9、西方墨點法量化（Western blotting quantitation）……………………….21
10、微陣列分析（Microarray analysis）……………………….…………….21
11、異位移植（Xenograft model）……………………………………………22
12、實驗數據統計分析（Statistical analysis）…………………………....…..23
陸、實驗結果………………………………………………………….………….24
1、AC009對於大腸癌細胞株的存活率抑制較AC007效果佳………..………...24
2、AC009會抑制大腸癌細胞之DNA生合成且造成G0/G1期阻滯.…..………..24
3、AC009會促使大腸癌細胞凋亡…………………………………...…………..25
4、AC009可有效抑制HCT116細胞之腫瘤生長……………….………………..26
5、miRNA microarray分析結果...……………………………………...……….27
柒、討論……………………………………………………………………….......28
捌、結論…………………………………………………………………………...32
玖、參考文獻……………………………………………………………….…….33

圖目錄

Table 1. AC007及AC009對於大腸癌細胞株之半數抑制濃度(IC50)……………38
Figure 1. AC009對於大腸癌細胞株的存活率抑制較AC007效果佳，且AC009對於HCT116細胞株有最佳存活率抑制效果……………………………………….39
Figure 2. AC009會抑制大腸癌細胞DNA合成且造成G0/G1期阻滯及 Sub G1期細胞凋亡………………...……………………………………………………....…..40
Figure 3. AC009會促使HCT116細胞凋亡………………………………………..42
Figure 4. AC009導致不顯著的細胞壞死…………………………………...……...44
Figure 5. AC009造成的大腸癌細胞凋亡和caspase、MAPK pathway及Bcl-2有關…………………………………………………………………………………….45
Figure 6. AC009可有效抑制HCT116細胞之腫瘤生長………………….……….49
Figure 7. miRNA microarray分析…………………………………………………..50
Figure 8. 牛樟芝萃取物AC009對於HCT116細胞之機制調控…………………51
Supplementary Figure 1. AC009對於MDM2及p53的蛋白調控………………...52

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