靈芝長久以來即被用在醫療用途上，特別是在東亞地區。在傳統中國醫學中，靈芝即被用來延長人的壽命和治療很多疾病，近代醫學更顯示出靈芝能夠治療癌症。靈芝的主要成分為多醣體和三萜類，多醣體主要是經由增強人體的免疫力因而達到抗癌的效果；三萜類主要是經由毒殺癌細胞而達到抗癌的效果。本實驗室主要使用松杉靈芝為實驗材料，之前的結果顯示松杉靈芝能對抗許多癌細胞，包括卵巢癌、乳癌、肺癌、皮膚癌、口腔癌及大腸直腸癌，本論文為了進一步探討松杉靈芝抑制口腔癌細胞的分子機轉，使用蛋白質體學技術。經由二維凝膠電泳及PDQuest分析軟體分析差異點，最後利用MALDI-TOF/TOF分析，及MASCOT資料庫比對，在HSC-3細胞株中鑑定出42個蛋白質差異點；在SCC-4細胞株中鑑定出33個蛋白質差異點。之後將這些蛋白質匯入GeneGo Meta CoreTM分析各蛋白質之間相互關係圖，在兩株不同的口腔癌細胞中找到相同的訊息傳遞路徑，另外也找到各別的訊息傳遞路徑。本論文對未來利用松杉靈芝萃取物抑制口腔癌細胞的生長提供了可能的訊息傳遞路徑。

Traditional Chinese Medicine (TCM) has been used to preserve the human vitality, to promote longevity, and for the prevention or treatment of a variety of diseases including cancer. Ganoderma, also known as Lingzhi, is a TCM and has been used for medicinal purposes for centuries. Among the eight common species of Ganoderma in Taiwan, Ganoderma tsugae (Gt) has been used in our study for fighting cancers. Our preliminary data showed that Gt extracts could inhibit the growth of human oral cancer cell lines, HSC-3 and SCC-4. To further unravel its molecular mechanism, a proteomics approach was applied. The proteomics experiments revealing the cancer cell growth inhibiting effect of Gt extracts was found to be associated with regulation of 42 proteins for HSC-3, and 33 proteins for SCC-4. All these proteins were subjected to the GeneGo Meta Core analysis to search for the possible molecular pathways. Interestingly, similar molecular pathways were found for these two oral cancer cell lines responding to Gt extracts treatment. These data suggest the possible molecular pathways leading to human oral cancer cell growth inhibition after Gt extracts treatment. Moreover, it may also be beneficial to the clinical treatment of human oral cancers.

中文摘要 i
英文摘要 ii
誌謝 iii
表目錄 vii
圖目錄 viii
縮寫表 ix
第一章：緒論 11
第一節、研究緣起與目的 11
第二節、靈芝(Ganoderma, Reishi, Lingzhi) 13
壹、靈芝概述 13
貳、靈芝之分類、分佈及其外形構造 13
參、靈芝之化學組成及其生理功能 14
肆、松杉靈芝(Ganoderma tsugae) 16
第三節、蛋白質體學 18
壹、蛋白質體學的定義 18
貳、蛋白質體學的研究方法 18
參、蛋白質鑑定技術 20
第二章：材料與方法 22
第一節、實驗材料 22
壹、細胞株 22
貳、松杉靈芝 23
參、化學試劑藥品 23
肆、主要器材儀器 26
第二節、實驗方法 27
壹、松杉靈芝乙醇萃取 27
貳、細胞株培養 28
參、測量松杉靈芝萃取物對細胞株的生長曲線 28
肆、測量松杉靈芝萃取物對細胞株的半數抑制濃度(IC50) 29
伍、測量松杉靈芝萃取物對細胞株的移動能力 (創傷癒合試驗 31
Wound closure assay) 31
陸、測量松杉靈芝萃取物對細胞株的生長週期 31
柒、二維凝膠電泳(2-Dimension Gel Electrophoresis) 32
捌、膠體內蛋白質水解(In Gel Digestion) 36
玖、介質輔助雷射脫附游離飛行時間式質譜儀(MALDI-TOF/TOF)及資料庫應用 37
拾、西方墨點法(Western Blotting) 38
第三章：結果 41
第一節、松杉靈芝萃取物對口腔癌細胞株的影響 41
壹、松杉靈芝萃取物對口腔癌細胞株的生長抑制曲線 41
貳、松杉靈芝萃取物對口腔癌細胞株的半數抑制濃度(IC50) 41
參、松杉靈芝萃取物對口腔癌細胞株形態的影響 41
肆、松杉靈芝萃取物對口腔癌細胞株移動能力的影響 42
伍、松杉靈芝萃取物對口腔癌細胞株生長週期的影響 42
陸、松杉靈芝萃取物對口腔癌細胞株生長週期蛋白質的影響 43
第二節、以二維電泳、質譜分析以及資料庫比對找出松杉靈芝萃取物對口腔癌細胞株蛋白質表現的影響 43
壹、以二維凝交電泳及介質輔助雷射脫附游離飛行時間式質譜分析投予松杉靈芝萃取物後之口腔癌細胞株蛋白質表現的影響 43
貳、彙整所有經二維電泳、質譜分析所得的蛋白質 44
第三節、應用生物資訊學推測松杉靈芝萃取物抑制口腔癌細胞株可能的訊息傳遞路徑 45
第四節、驗證松杉靈芝萃取物抑制口腔癌細胞株的訊息傳遞路徑 46
第四章：討論 48
第一節、蛋白質體學研究方法討論 48
第二節、相同的訊息傳遞路徑圖 49
第三節、各別的訊息傳遞路徑圖 53
第五章：結論 57
第六章：參考文獻 59
第七章：圖表 65
第一節、表 65
第二節、圖 71
附錄 89
附錄一、台灣地區常見野生靈芝一覽表(許瑞祥教授發表) 89
附錄二、靈芝的主要成分及其療效 90
附錄三、細胞株培養方法 91
附錄四、流式細胞儀使用之緩衝液配置 94
附錄五、二維凝膠電泳及膠體內蛋白質水解試劑及緩衝液配置 95
附錄六、二維凝膠電泳條件 98
附錄七、GeneGO Meta CoreTM (version 5.1 build 16271) signaling networks legend 99
附錄八、西方墨點法試劑及緩衝液配置 102

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