大腸癌在全世界的癌症發生率排名第三高。在台灣衛福部的統計資料顯示，目前國人癌症致死率大腸癌排第三名。
AMP-activate protein kinase (AMPK)是維持細胞能量平衡之重要調節因子，近年來研究發現，AMPK 活化不只可以當能量調解的角色，也可以抑制細胞的生長；AMPK 活化可以減緩癌細胞的生長。
常用的大腸直腸癌化學治療藥物包括5-Fluorouracil (5-FU)、Oxaliplatin (OXR)等，已經有研究提出，癌細胞對於5-FU或OXR產生抗藥性的機制，這些機制包括了增強DNA修復的能力並阻止細胞凋亡產生，而DNA修補蛋白如MSH2或是XRCC1表現量較高時，對於會造成細胞毒性的藥物會產生抗藥性。
本論文主要探討在大腸癌細胞株中，AMPK活化劑AICAR增強5-FU或OXR所誘導的細胞毒性作用的影響。發現將大腸癌細胞株處理5-FU或OXR，會提高Akt的磷酸化，並增加MSH2及XRCC1表現，若將AICAR和這兩個化療藥物合併處理，會協力降低MSH2及XRCC1蛋白質表現量。期望在未來大腸癌的治療上能提供更好的方法與研究方向。

關鍵字: 大腸癌、AMPK、5-FU、OXR、MSH2、XRCC1、 AICAR

Colorectal cancer is the third most commonly diagnosed cancer in the world and the third leading cause of cancer mortality in Taiwan.
AMP-activate protein kinase (AMPK) is the important mediation energy sensing factor in human cell. The recent studies found that AMPK is not only a regulator of energy but also an inhibitor of cell growth. Activation of AMPK could slow down growth of cancer cell.
Chemotherapy has been the mainstay approach for patients with advanced colorectal cancer. 5-Fluorouracil (5-FU) and Oxaliplatin (OXR) are the active anticancer agents available to treat advanced colorectal cancer. In eukaryotes, MSH2 or XRCC1 is the key enzymes of DNA repair systems. High level of MSH2 or XRCC1 expression is resistant to cytotoxic agents.
In this proposal, we investigated the effect of AMPK activator AICAR on enhancing the 5-FU or OXR-induced cytotoxicity in colorectal cancer (CRC) cells.
Our preliminary results showed that exposure of human CRC cell line HCT-116 to 5-FU or OXR could increase protein levels of phosphorylated Akt, and increased expression of DNA repair genes MSH2 and XRCC1.
We also observed that the combined treatment of AICAR and these two chemotherapy drugs induced synergistic reduction protein levels of MSH2 and XRCC1.
Our study will further investigate the molecular mechanisms on regulation of MSH2 and XRCC1 expression by AMPK agonist to enhance cytotoxicity of chemotherapy drugs in human colorectal cancer cells.

Key words : Colorectal cancer , AMPK , 5-FU , OXR , MSH2 , XRCC1, AICAR

中文摘要 ………..…………………………………….. Ⅰ
英文摘要 ..…………………………………………….. Ⅱ
致謝 ..…………………………………………….. Ⅲ
目錄 …..………………………………………….. Ⅳ
圖目錄 ………………..…………………………….. Ⅶ
1. 大腸直腸癌 (Colorectal Cancer) 1
1-1大腸直腸癌成因 1
(The cause of Colorectal Cancer
1-2大腸直腸癌的病理特徵 2
(pathophysiology of Colorectal Cancer)
1-3大腸直腸癌的治療 3
(Treament of Colorectal Cancer)
2. 化療藥物Oxaliplatin (OXR)與5-fluororacil (5-FU) 3
2-1 Oxaliplatin (OXR) 4
2-2 5-fluororacil (5-FU) 4
3. DNA修補(DNA repair) 5
3-1 DNA修補方式 (DNA repair manner) 5
3-2 DNA錯誤配對修復系統 5
(DNA mismatch repair system, MMR )
3-3 鹼基切除修復(Base excision repair, BER) 6
4. AMP-activated protein kinase (AMPK) 7
5. p38及Akt訊息傳遞路徑(p38 & Akt pathway) 8
6. PI3K/Akt pathway 9
7. 研究動機 (Research Motive) 9
第二章 材料與方法 (Materials and Methods)
2-1 實驗材料與試劑 10
A.細胞株 10
B.藥品及材料 10
2-1-2實驗材料配方 14
A.細胞培養 14
B.細胞存活率分析 14
C.基因表現量分析 15
D.蛋白表現量分析 15
2-2 實驗器材 16
A.細胞培養 16
B.細胞存活率分析 16
C.基因表現量分析 16
D.蛋白表現量分析 16
2-3實驗方法 17
A.細胞培養 17
B.細胞存活率分析 18
C.基因表現量分析 19
D.蛋白表現量分析 25
第三章 結果 (Results)
3-1 OXR與5-FU抑制大腸癌細胞株HCT-116的生長 28
3-2 OXR與5-FU刺激HCT-116細胞中DNA修補基因MSH2及XRCC1表現 28
3-3 利用MAPKs抑制劑和PI3K/Akt抑制劑，探討OXR及5-FU誘導MSH2和
XRCC1基因表現 29
3-4 MAPKs抑制劑處理HCT-116細胞，OXR和5-FU對細胞存活率的表現 29
3-5 OXR與5-FU刺激HCT-116細胞中MSH2和XRCC1蛋白表現 30
3.6 AMPK活化劑AICAR與OXR和5-FU協同作用抑制HCT-116細胞生長 29
3.7 AMPK活化劑AICAR與OXR和5-FU協同作用下抑制HCT-116細胞中MSH2及XRCC1基因表現 29
第四章 討論 (Disscussion) 32
結論(Conclusions) 38
參考文獻 39
圖 47
附錄 60

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