靈芝自古即被”神農本草經 ”與”本草綱目”列為上品中草藥，具有延年益壽功能。近年來亦有相關文獻報導靈芝具有抗老化與抗腫瘤效果。另外先前的研究指出腫瘤初始形成及免疫耐受性的產生與吲哚胺 2,3-雙氧化酶 (IDO) 蛋白有很大的相關性，IDO藉由催化必需胺基酸色胺酸而抑制了對色胺酸短缺極為敏感的T細胞增生，造成T細胞生長受到抑制。此外，第二型人類表皮生長因子受體 (HER2) 與癌症的惡性度息息相關，包括腫瘤的增生、轉移、侵襲能力，造成癌症臨床預後效果不佳的狀況。因此，本論文探討靈芝是否可藉由抑制HER2誘導產生的IDO進而抑制癌細胞的免疫逃脫現象。結果發現靈芝乙醇萃取物在HER2高度表現細胞株中具有濃度依賴性的抑制HER2的表現，此種抑制現象在RNA及蛋白質的層次上皆有相同的效果。HER2受抑制後，進而使IDO基因及其蛋白表現也受到抑制。綜合以上結果顯示靈芝乙醇萃取物具有抗癌效果，同時藉由抑制IDO的基因表現，顯示也可能具有增強免疫能力的作用，是一個極具有開發潛力的中草藥。

Lingzhi has been classified as one of the best grade of traditional Chinese medicines (TCMs) by the Chinese ancient medical encyclopedia, ”Shen-Nong-Ben-Cao-Jing” and “Ben-Cao-Gang-Mu”. Recent reports have shown that Lingzhi, including Ganoderma tsugae, has anti-tumor and antioxidative properties. On the other hand, the protein indoleamide 2,3-dioxygenase (IDO) has been demonstrated to be closely involved in tumor formation and immune tolerance. IDO catalyzes the metabolism of the essential amino acid “tryptophan” resulting in blocking T cell proliferation. While the aberrant expression of human epithelial growth factor receptor 2 (HER2; ErbB2) is known to be associated with cancer malignancy, including tumor progression, tumor metastasis, and invasive abilities, leading to poor clinical outcomes. In this study, we reveal that the Ganoderma tsugae ethanol extract (GTEE) can be used to down-regulate the expression of HER2 in HER2-overexpression cancer cell lines. Furthermore, the differential suppression of HER2 is in response to the inhibition of IDO along with a dose-dependent increase of GTEE. These results provide that GTEE may be a potential TCM for blocking tumor immune escape and enhancing anti-tumor effects.

中文摘要 I
英文摘要 II
誌謝 III
目錄 V
研究緣起 1
第一章 文獻回顧 3
第一節 吲哚胺 2,3-雙氧化酶 (Indoleamine 2,3-dioxygenase; IDO) 3
一、 IDO蛋白質的功能與結構 3
二、 IDO的免疫耐受性與癌症之關係 4
第二節 人類表皮生長因子接受器第二型 5
一、HER2的功能與結構 5
二、HER2的訊息傳遞路徑 6
第三節 靈芝 7
一、 靈芝概述 7
二、 靈芝醫療相關研究 8
三、 實驗用松杉靈芝(Ganoderma tsugae)來源 10
第二章 材料與方法 11
第一節 藥品與試劑 11
第二節 主要器材與儀器 14
第三節 細胞株 15
第四節 實驗方法 15
壹、細胞培養 15
貳、靈芝萃取 18
参、西方點墨法 (Western blotting) 19
肆、核醣核酸的萃取 (Isolation of total RNA) 21
伍、DNA轉染 (Transfection) 23
陸、冷光測試啟動子活性 (Luciferase assay) 23
柒、細胞存活分析 (MTT assay) 24
捌、統計分析 (Statistical Analysis) 25
第三章 結果 26
第一節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞株的影響 26
1、 靈芝乙醇萃取物對SKOV-3卵巢癌細胞株的半數抑制
濃度 (IC50) 26
2、 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2乳癌細胞株形態的影響 26
第二節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2
乳癌細胞中的HER2蛋白質表現量的影響 27
第三節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2
乳癌細胞中的HER2蛋白質表現量的影響 27
第四節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞及MCF7/HER2
乳癌細胞株中IDO mRNA表現量的影響 28
第五節 靈芝乙醇萃取物對SKOV-3卵巢癌細胞株中蛋白質穩
定度的影響 28
第六節 靈芝乙醇萃取物對SKBR-3及MCF7/HER2乳癌細胞
株啟動子活性影響 29
第七節 靈芝乙醇萃取物是藉由何種路徑影響卵巢癌細胞SKOV-3及乳癌細胞MCF7/HER2中的HER2及IDO 30
第四章 討論 31
第五章 結論 36
第六章 參考文獻 37
第七章 圖表 42

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