臺灣博碩士論文加值系統

惡性腫瘤已多年蟬聯台灣十大死因之首，其中大腸直腸癌是第三常見癌症死因亦是癌症發生人數最多的癌症。即便目前治療方法多種，但癌細胞高度的侵襲及轉移能力，是導致大腸癌患者預後不良主因之一。已有研究指出非類固醇消炎止痛藥物，有抑制癌細胞生長、促進細胞凋亡和抑制癌細胞轉移的能力。Parecoxib是目前唯一注射型的選擇性第二型環氧酶抑制劑的非類固醇消炎止痛藥物。目前研究大多發現選擇性第二型環氧酶抑制劑抑癌效果與其抑制第二型環氧酶有關。但對於其無表現第二型環氧酶的癌細胞是否仍具有抑制癌細胞轉移的機制並無詳盡探討。因此，本實驗首先要來探討parecoxib對於無表現第二型環氧酶大腸癌細胞DLD-1侵犯轉移能力的影響，然後再用西方墨點法分析其抑制癌細胞之機制。實驗結果發現對於無表現第二型環氧酶的大腸癌細胞株DLD-1，parecoxib會透過抑制Akt磷酸化來影響β-catenin訊息傳遞的表現，進而減少上皮間質轉化來抑制大腸癌細胞DLD-1轉移。本篇有助於parecoxib作為合併治療大腸癌轉移相關藥物的輔助治療。

Colorectal cancer is the third leading cause of cancer death in Taiwan. Although current treatments involving a combination of surgical resection, radiation, and chemotherapy have increased the patient’s five-year survival, metastasis is a major capability of cancer cells that causes poor prognosis, recurrence and even death. Epidemiological and clinical studies suggest the nonsteroidal anti-inflammatory drugs (NSAIDs) represent an effective class of compounds for the prevention of colon cancer. Parecoxib is one of NSAID and is the only parenterally administered selective cyclooxygenase (COX)-2 inhibitor. The aim of this study was to investigate whether parecoxib inhibits metastasis of non-COX-2 DLD-1 human colon cancer cells and the underlying mechanism. Cell viability assay by MTT method revealed that parecoxib did not affect viability of DLD-1 cells. Subsequently, parecoxib significantly inhibited the cell motility of DLD-1 cells by transwell migration assay. Moreover, we also show that parecoxib attenuated phosphorylation of Akt, β-catenin, the mesenchymal marker-vimentin and upregulated GSK3β, the epithelial marker-E-cadherin. These findings suggest parecoxib can inhibit epithelial-mesenchymal transition (EMT) and metastasis in human colon cancer cells via down regulation of β-catenin. These results suggest that parecoxib is a good candidate for a combination treatment against human colon cancer metastasis.

誌謝....................................................I
中文摘要................................................II
英文摘要...............................................III
目錄...................................................IV
圖表目錄..............................................VIII
縮寫表.................................................IX
第一章 緒論.............................................1
第一節 大腸癌...........................................1
第二節 癌症轉移機制......................................3
一、 癌症轉移步驟........................................3
二、 癌細胞轉移運用的多種策略.............................3
第三節 NSAIDS和大腸癌的關係..............................7
第四節 PARECOXIB........................................9
第五節 上皮間質轉化(EMT)和癌症轉移.......................11
一、EMT的概念...........................................11
二、EMT和癌症轉移的關係..................................13
第六節 WNT/β-CATENIN訊息傳遞路徑........................14
第七節 MACC1/C-MET/AKT訊息傳遞路徑......................16
第八節 MITOGEN-ACTIVATION PROTEIN KINASES (MAPK)訊息傳遞路
徑...............................................17
第二章 研究動機及目的....................................19
第三章 材料與方法.......................................20
第一節 實驗材料.........................................20
一、細胞株 (Cell line).................................20
二、藥品...............................................20
三、西方墨點法所使用的抗體...............................21
四、實驗儀器............................................22
五、軟體分析............................................23
第二節 實驗方法.........................................23
一、細胞培養 (Cell culture) -貼壁細胞....................23
二、細胞活性測試 (MTT assay)測定細胞存活率與細胞毒性......24
三、細胞遷移實驗 (Cell migration assay).................25
四、總蛋白萃取 (Total Protein Extraction)...............25
五、核蛋白萃取 (Nuclear Protein Extraction).............26
六、蛋白定量分析 (Quantitative of protein concentration)27
七、西方墨點法 (Western blot)...........................27
八、穩定表現 Akt phosphorylation overexpression的細胞之建立
.................................................. 30
九、統計分析............................................32
第三節 試劑配方.........................................32
一、 DLD-1細胞培養液....................................32
二、 10X PBS製備........................................33
三、 抽取細胞內總蛋白質之試劑配法.........................33
四、 抽取細胞內核蛋白試劑配法............................33
五、 蛋白質定量.........................................34
六、 西方墨點法所需之試劑配法............................35
七、 穩定表現 Akt phosphorylation overexpression 的細胞之建
立................................................38
第四章 結果.............................................39
第一節 觀察PARECOXIB對於大腸癌細胞DLD-1細胞存活率之影響...39
第二節 PARECOXIB 對於大腸癌細胞DLD-1之細胞轉移作用的影響..39
第三節 PARECOXIB抑制DLD-1細胞轉移之機制探討..............40
一、Parecoxib影響大腸癌細胞DLD-1上皮間質化相關蛋白的表現..40
二、Parecoxib會抑制GSK3下游-catenin的表現.............40
三、Parecoxib 會抑制MACC1/c-Met/AKT訊息傳遞路徑..........41
四、探討Parecoxib 對於MAPK pathway的影響................42
五、探討Parecoxib 主要是藉由抑制Akt的磷酸化來抑制DLD-1轉移43
第五章 討論.............................................44
第六章 結論.............................................49
第七章 參考文獻.........................................50
第八章 圖..............................................61

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