鼻咽癌(Nasopharyngeal carcinoma, NPC)是一種頭頸部癌症，在全世界並不常發生，但是在台灣、中國的南方(如：福建、廣東一帶)、阿拉斯加、格陵蘭及東南亞等地卻有很高的發生率。鯽魚膽(Pluchea indica (L.) Less)是一種常見的中草藥，分布於熱帶國家的濱海或鹽澤地區，此植物傳統上具有抗發炎、解熱及收斂的效果。本研究的目的是為了證實鯽魚膽根部乙醇萃取物(ethanolic extract of Pluchea indica root)對於鼻咽癌細胞株生長的影響及其抗癌的相關分子機制。本研究中，細胞的存活率利用WST-1分析進行實驗，並利用群落生長分析(colony formation assay)研究鯽魚膽根部乙醇萃取物對鼻咽癌細胞增生的影響。鯽魚膽根部乙醇萃取物對鼻咽癌細胞移行(cell migration)的影響則分別利用體外傷口癒合分析(wound healing assay)及細胞穿膜試驗(transwell assay)進行分析。鯽魚膽根部乙醇萃取物對細胞週期的影響，利用流式細胞儀(flow cytometry)決定，而細胞凋亡所形成的DNA片段(DNA fragmentation)則採用末端去氧核苷酸轉移酶引導的dUTP 缺口分析(Tdt-mediated dUTP nick end labelling (TUNEL) assay)偵測。凋亡相關的基因表現，利用及時定量聚合酶連鎖反應(RT-PCR)及西方墨點法(western blotting)進行分析。研究結果顯示鯽魚膽根部乙醇萃取物能有效的抑制NPC-TW01及NPC-TW04兩株鼻咽癌細胞的存活率及增生，此抑制效果具有藥物劑量的依賴性，且NPC-TW04細胞對鯽魚膽根部乙醇萃取物具有更高的敏感性。實驗結果也證實鯽魚膽根部乙醇萃取物可抑制鼻咽癌細胞的移行，且隨著鯽魚膽根部乙醇萃取物的劑量增加，位於sub-G1期的鼻咽癌細胞比例也會隨之增加。在凋亡的分子機制方面，鯽魚膽根部乙醇萃取物增加p53 mRNA及蛋白質的表現，同時也增加BAX的表現及抑制Bcl-2表現，此外，鯽魚膽根部乙醇萃取物也增加鼻咽癌細胞中Bax/Bcl-2的比值，藉由以上這些相關的機制，進而抑制鼻咽癌細胞的增生。本研究的結果顯示在開發抗鼻咽癌新藥時，可以考慮由鯽魚膽的活性萃取物著手。

Nasopharyngeal carcinoma (NPC) is a head and neck cancer that rarely occurs in most parts of the world. In contrast, the highest incidence was found in the southern Chinese population of Guangdong, Inuits of Alaska, and native Greenlanders. Pluchea indica (L.) Less is a well-known indigenous medicinal plants that grows in littoral areas of tropical regions of countries such as Malaysia, Indonesia, Australia, Taiwan, India, and Mexico. The plants have been traditionally used as astringent and antipyretic. The present study is to investigate the anti-cancer effect of the ethanolic extract of Pluchea indica (L.) Less root (PIRE) and its possible molecular mechanism in human nasopharyngeal carcinoma (NPC) cells. Cell viability were measured by WST-1 experiments and Colony formation assay. Cell migration were measured by wound healing assay and transwell assay in vitro, respectively. The sub-G1 ratio was examined using flow cytometry and the extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling (TUNEL) assay. The expression of apoptosis-related proteins were assessed by Western blotting. We found that PIRE could strongly inhibit cell viability of NPC-TW01 and NPC-TW04 cells in dose-dependent manner; however, NPC-TW04 cells showed more susceptible to the treatment. Furthermore, cell migrations were also suppressed with exposure to PIRE. This study also found that PIRE could significantly increase the occurrence of the sub-G1 phase and the extent of DNA fragmentation in a dose-dependent manner, which showed that, PIRE significantly increased apoptosis in NPC cells. The apoptotic process triggered by PIRE involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. In the meantime, we also observed that PIRE could increase the ratios of Bax/Bcl-2 protein levels and up-regulation of the p53 protein in a concentration-dependent manner. Therefore, PIRE could induce the apoptosis signaling pathway in NPC cells by activation of p53 and by regulation of apoptosis-related proteins. In summary, this study suggest that Pluchea indica could be further investigated as a new alternative chemotherapeutic agent for NPC.

論文審定書 ⅰ
誌謝 ⅱ
中文摘要 ⅲ
英文摘要 ⅴ
壹、緒論 1
一、前言 1
二、鼻咽癌病因 2
三、鼻咽癌分子機轉 5
四、鼻咽癌病理學與臨床表徵 7
五、鼻咽癌治療 7
六、鯽魚膽 8
七、細胞凋亡 9
貳、實驗目的 15
參、材料與方法 16
一、鯽魚膽根部乙醇萃取物之製備 16
二、初步化學成份分析 16
三、細胞培養 18
四、細胞增值及存活分析 20
五、細胞群落形成分析 20
六、體外傷口癒合分析 21
七、細胞移行分析 21
八、流式細胞儀分析 22
九、TUNEL staining 23
十、即時定量聚合酶連鎖反應 24
十一、西方墨點法 26
十二、數據統計分析 28
肆、結果 29
伍、討論 35
陸、參考文獻 41
柒、圖表 61
捌、附錄 79

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