猴頭素A是從猴頭菇菌絲體分離出的活性成分，已被證實具有抗癌作用。但猴頭素A抑制人類胃癌細胞生長的機制尚未很明確。因此，我們想探討猴頭素A如何影響誘導胃癌細胞細胞凋亡和抑制胃癌細胞侵襲狀態和生長相關的分子機制。我們運用MTT assay 和Boyden chamber assay來確定經猴頭素A處理後對胃癌細胞增殖能力和侵襲力的影響；蛋白質體學方法和蛋白質印跡分析提供的資訊用於鑑定猴頭素A處理後的差異表達蛋白質及其作用於凋亡的作用機制和猴頭素A作用的靶蛋白。我們的研究結果發現，猴頭素A處理TSGH 9201細胞後會增加細胞毒性和活性氧的產生以及細胞增殖和侵襲性的降低。而且猴頭素A處理TSGH 9201細胞會導致caspase 8，caspase 3和caspase 9的活化，cytochrome c的釋放和TRAIL的表達；而抗凋亡因數Bcl-2和Bcl-XL的表達顯著降低。猴頭素A誘導凋亡伴隨著FAK / AKT / p70S6K和PAK1途徑的持續磷酸化，以及ROS的產生。此外，通過啟動FAK / AKT / p70S6K和PAK1信號通路，猴頭素A誘導凋亡可能涉及14-3-3 protein sigma (1433S)和microtubule-associated tumor suppressor candidate 2（MTUS2）的差異表達，誘導TSGH 9201胃癌細胞凋亡。

Erinacine A, a major active component of a diterpenoid derivative isolated from Hericium erinaceus mycelium, has been demonstrated to exert anticancer effects. Herein, we present an investigation of the molecular mechanism of erinacine A inhibition associated with gastric cancer cells' aggressive status and growth. The cell viability and boyden chamber assays were applied to determine the effects of erinacine A treatment on the proliferation capacity and invasiveness of gastric cancer cells. A proteomic approach and western blot analyses provided information were used to purify and identify the differentially expressed proteins following erinacine A treatment and the mechanism of its action in apoptotic and the targets of erinacine A. Our results demonstrate that erinacine A treatment of TSGH 9201 cells increased cell cytotoxicity and reactive oxygen species (ROS) production as well as decreased cell proliferation and invasiveness. Treatment of TSGH 9201 cells with erinacine A resulted in activation of the caspase 8, caspase 3 and caspase 9, release of cytochrome c and the expression of TRAIL; whereas, the expression of anti-apoptosis factor Bcl-2 and Bcl-XL were decreased markedly. Erinacine A induction of apoptosis was accompanied by sustained phosphorylation of FAK/AKT/p70S6K and PAK1 pathways, as well as generation of the ROS. Furthermore, the induction of apoptosis and anti-invasion properties by erinacine A could be involved the differential expression of 14-3-3 protein sigma (1433S) and microtubule-associated tumor suppressor candidate 2 (MTUS2), with activation of the FAK/AKT/p70S6K and PAK1 signalling pathways. These results leads us to speculate that erinacine A may play an apoptotic cascade in TSGH 9201 cells by activation of the FAK/AKT/p70S6K and PAK1 pathway and induction of ROS production, providing a new mechanism for erinacine A-induced anti-aggressiveness and cell death in human gastric cancer cells.

審定書……………………………………………………………………...…………….i
致謝……………………………………………………………………………………...ii
中文摘要……………………………………………..………………………………....iii
Abstract………………………………………………………………………………...iv
目錄……………………………………………………………………………………...v
圖次………………………………………………………….…………………….…....ix
表次……………………………………………………………………….……………..x
第一章 緒論………………………………………………………………………...…..1
1.1 前言……………………………………………….……..………….…….......1
1.2 胃癌介紹………………………………………….…………..…………........1
1.2.1 胃癌之發生率以及死亡率排行……………………….…………..........1
1.2.2 胃癌種類……………………………………………………….…..........2
1.2.2.1 腸道型………………………………………....……...…..............2
1.2.2.2 瀰漫型…………………………………..……....…….…..............2
1.2.3 胃癌分期……………………………….....………………...……..........3
1.2.4 危險因數………………………………..……….……...……..…..........4
1.2.4.1 幽門螺旋桿菌………………………..……...……..................4
1.2.4.2 飲食生活習慣………………………..………...…..................4
1.2.4.3 遺傳……………………………………..……….....................5
1.2.4.4 胃切除手術……………………………..…...……..................5
1.2.2.5 其他因素…………………………………..…...…..................5
1.2.5 胃癌的診斷………………………………………...………..….............5
1.2.6 胃癌治療方式………………………………………...………...............6
1.3 研究動機與目的…………………………………………………..……….....6
第二章 文獻探討……………………………………………………………………….7
2.1 猴頭菇萃取之活性成分Erinacines A………………………..….…...…......7
2.1.1 Erinacines A的化學特性………………………………..……..............7
2.1.1.1 學名………………………………………………………..............7
2.1.1.2 結構式………………………………………....…...……..............7
2.1.1.3 分子式…………………………….…………...……...…..............7
2.1.1.4 猴頭菌代謝產物與生理活性功能……………..……...…............7
2.2 細胞凋亡作用…………………….………………………..…...……….......8
2.2.1 細胞凋亡的特徵……………………………………………….….........8
2.2.2 細胞凋亡與細胞壞死之區別……………………………………..........9
2.2.3 細胞凋亡的調控機制……………………………………………..........9
2.2.3.1 外在途徑………………………………………..………..............9
2.2.3.2 內在途徑………………………….………….………................10
2.2.3.2.1 ROS誘導細胞凋亡……………...………......................11
2.3 蛋白質體學…………………….……..…………………..….…….............11
2.3.1 二維膠體電泳…………………………………..………..…...….........11
第三章 材料與方法…………………………………………………………………...13
3.1 實驗材料…………………….………………………..…...……..................13
3.1.1 細胞株………………………………………………………….….........13
3.1.2 化學試劑和抗體……………………………………………….….........13
3.2 實驗方法…………………….………………………..…...…….................13
3.2.1 猴頭菇活性成分Erinacines A的萃取與分析…………...……...........13
3.2.2 細胞培養……………………………………………...…………..........14
3.2.3 繼代培養………………………………………………...………..........14
3.2.4 細胞冷凍保存……………………………………………...……..........14
3.2.5 細胞活化……………………………………………...…………..........15
3.2.6 DAPI (4',6-diamidino-2-phenylindole)染色…………...……….........15
3.2.7 Annexin V/PI雙重染色…………………………………...….…..........15
3.2.8 細胞存活率分析…………………..………………….……....…..........16
3.2.9 活性氧化物質分析……………………...………………………..........16
3.2.10 細胞轉移能力分析…………………….………………….….............16
3.2.11 蛋白質濃度測定……………………….…...……………….…..........17
3.2.12西方墨點法……………………………..……...…………….…..........17
3.2.13 二維差異電泳分析與影像分析…………….…...………….…..........18
3.2.13.1 猴頭菇菌絲體蛋白質萃取液之製備…...……….….................18
3.2.13.2 蛋白質定量………………………………......…....…...............18
3.2.13.3 蛋白質等電點聚焦電泳法…………………...….….................19
3.2.13.4 第二維電泳…………………………….………...….................19
3.2.13.5 銀染…………………………………………..….…..................20
3.2.13.6 二維膠電泳影像處理與分析………………….…....................20
3.2.13.7 使用MALDI-TOF/ TOF鑒定蛋白質………….…....................20
3.3 統計分析…………………….………………………..…...……..................21
第四章 實驗結果…………………………………………………………………...…..22
4.1. Erinacines A處理TSGH 9201細胞產生細胞型態的變化…………….......22
4.2 Erinacines A能誘導TSGH 9201細胞Apoptosis……………....……..........22
4.3 Erinacines A能抑制TSGH 9201細胞的轉移能力……………...................23
4.4 Erinacines A能誘導TSGH 9201細胞產生ROS………..……….................23
4.5 Erinacines A誘導TSGH 9201細胞引發凋亡信號傳遞路徑之探討…................24
4.6 利用二維膠體電泳來檢測Erinacine A處理TSGH 9201蛋白質的表現情形.......24
4.7 利用Western blot再次確認二維膠體電泳鑑定到的蛋白質………….................25
4.8 Erinacine A誘導胃癌細胞FAK / AKT / p70S6K和PAK1磷酸化表現..........…...26
第五章 討論…..……………………………………………………………………....…27
參考文獻………………………………………………………..…………………….....30

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