肺癌是佔癌症死亡最重要原因並且目前的化學治療藥物療效易有抗藥性發生。腫瘤壞死因子相關凋亡誘導配體（TRAIL）是具潛力抗癌藥物，因為它可以選擇性地誘發癌細胞凋亡，但不會影響正常細胞。然而WIN 55,212-2，一種人工合成的CB1/CB2大麻酚受體激化物，能在腫瘤細胞株誘導細胞凋亡。然而，在WIN55, 212-2合併TRAIL作用所介導的細胞凋亡的分子機制尚未在肺癌中被證實。在本研究中，我們表明，WIN55, 212-2中以劑量依賴的方式誘導細胞死亡，負調節誘餌受體 (DcR2、DcR3)，並大幅正向調節死亡受體 (DR5)的表現。在肺腺癌A549細胞中，WIN55,212-2介導TRAIL死亡受體DR5的上調，似乎透過CHOP基因表現增加來調節DR5基因的表現，此外，WIN55,212-2負調節細胞存活相關的基因 (Bcl-2、Mcl-1與Survivin)表現。此外，本實驗室構建DR5啟動子螢光素酶質體包含第一內含子區域內涵蓋p53、CHOP 和 NF-κB DNA等結合位點。DR5啟動子活性分析顯示，WIN55,212-2促進DR5基因轉錄現象。因此，這些實驗數據證明，WIN55, 212-2藉由透過上調DR5能強化TRAIL引發的細胞凋亡。綜合以上，這些結果表明，WIN55, 212-2 / TRAIL合併使用來防止肺癌細胞生長發展具有密切的相關。

Lung cancer is a major cause of cancer-related death and is resistant to current chemotherapy agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential target for cancer therapy as it selectively kills cancer cells but is non-toxic to normal cells. However, many cancer cells are resistant to TRAIL-induced apoptosis and its mechanism remains unclear. WIN 55,212-2, a synthetic CB1/CB2 receptor agonist, has proven to induce apoptosis in a variety of cancer cells. The aim of this study was to analyze the improvement of anticancer effect of TRAIL by WIN55,212-2 in lung cancer cells. Our results showed that WIN55,212-2 induced cell death in a dose-dependent manner in A549 cells.WIN55,212-2 could down-regulated the expression of decoy Receptors (DcR2, DcR3) and up-regulated the expression of death Receptor5 (DR5). The WIN55,212-2 induced apoptosis in A549 cells was partially regulated by CHOP-mediated DR5 up-regulation. Moreover, WIN55,212-2 treatment decreased the survival factor including Bcl-2, Mcl-1 and Survivin. Furthermore, a luciferase reporter assay revealed that the promoter activity of DR5 including the p53、CHOP and NF-κB DNA-binding site was induced upon WIN55,212-2 treatment. These data indicated that WIN55,212-2 can potentiate TRAIL-induced apoptosis through up-regulation of DR5. Taken together, these results suggest that the WIN55,212-2/TRAIL combination is closely related to prevent the progression of the lung cancer cells.

論文審定書 ……………………………………………………i
致謝 ……………………………………………………………… ii
中文摘要 ………………………………………………………… iii
英文摘要 ………………………………………………………… iv
目錄 ……………………………………………………………… v
圖目錄 ……………………………………………………… …x
縮寫檢索表 ……………………………………………………xi
第一章 緒論…………………………………………………… 1
第一節 肺癌…………………………………………………… 1
一 肺癌之簡介………………………………………………… 1
二 肺癌之流行病學…………………………………………… 1
三 肺癌之成因………………………………………………… 2
四 肺癌之分類………………………………………………… 2
五 肺癌之治療………………………………………………… 3
六 肺癌之標靶藥物…………………………………………… 6
第二節 大麻酚之相關介紹…………………………………… 6
一 大麻酚之基本介紹………………………………………… 6
二 大麻酚細胞表面接受器之介紹…………………………… 7
三 大麻酚受體激化物之分類………………………………… 8
四 大麻酚受體激化物與癌症之關係………………………… 8
第三節 細胞凋亡……………………………………………… 9
一 細胞凋亡之介紹…………………………………………… 9
二 細胞凋亡蛋白之介紹……………………………………… 10
三 細胞凋亡路徑之介紹……………………………………… 11
第四節 TRAIL與TRAIL receptor之關係…………………… 12
研究目的與動機…………………………………………… 15
實驗設計…………………………………………………… 16
第二章 實驗材料與方法……………………………………… 17
第一節 實驗材料……………………………………………… 17
一 細胞株……………………………………………………… 17
二 實驗藥品…………………………………………………… 17
三 儀器設備與耗材…………………………………………… 20
第二節 實驗方法……………………………………………… 22
一 細胞培養…………………………………………………… 22
二 藥品處理…………………………………………………… 22
三 MTT細胞存活試驗……………………………………… 22
四 細胞凋亡DNA片段化分析……………………………… 23
五 DAPI染色………………………………………………… 24
六 蛋白質標準品減量線製作………………………………… 24
七 細胞蛋白質萃取…………………………………………… 24
八 細胞蛋白質濃度測定與變性……………………………… 25
九 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳…………………… 25
十 西方墨點法………………………………………………… 25
十一 細胞RNA萃取…………………………………………… 26
十二 反轉錄聚合酶鏈反應……………………………………… 26
十三 轉染分析…………………………………………………… 27
十四 報導基因分析……………………………………………… 27
十五 統計分析…………………………………………………… 28
第三章 實驗結果……………………………………………… 29
第一節 大麻酚受體激化物WIN55,212-2與TRAIL對肺腺癌
A549細胞的生長抑制情況……………………………… 29
第二節 大麻酚受體激化物WIN55,212-2與TRAIL對肺腺癌
A549細胞形態的影響…………………………………… 30
第三節 大麻酚受體激化物WIN55,212-2與TRAIL對肺腺癌
A549細胞凋亡之影響…………………………………… 30
第四節 大麻酚受體激化物WIN55,212-2作用下DR5、CHOP
基因於肺腺癌A549細胞的變化………………………… 30
第五節 大麻酚受體激化物WIN55,212-2對肺腺癌A549細胞
內死亡受體DR5與誘餌受體DcR2、DcR3表現量之影響………………………………………………………………… 31
第六節 大麻酚受體激化物WIN55,212-2對肺腺癌A549細胞內
凋亡基因表現量之影響…………………………………… 32
第七節 大麻酚受體激化物WIN55,212-2與TRAIL作用下DR5、
CHOP表現量之影響……………………………………… 32
第八節 大麻酚受體激化物WIN55,212-2與TRAIL作用下與抗氧
化劑NAC共同處理對肺腺癌A549細胞內CHOP表現量之影響………………………………………………………………… 33
第九節 大麻酚受體激化物WIN55,212-2與TRAIL作用下對肺腺
癌A549細胞內凋亡相關蛋白質之影響………………… 33
第十節 大麻酚受體激化物WIN55,212-2作用下對DR5啟動子
表現之影響………………………………………………… 34
第十一節 大麻酚受體激化物WIN55,212-2作用下對轉錄因子
NF-κB表現之影響……………………………………… 34
第四章 結論與討論…………………………………………… 35
參考文獻 ………………………………………………………… 38
附圖 ……………………………………………………………… 42
附錄 ……………………………………………………………… 52

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