臺灣博碩士論文加值系統

中文摘要
樟芝又稱牛樟芝或牛樟菇，學名為Antrodia camphorata，是台灣特有一種真菌，僅生長於台灣特有之牛樟樹，生長緩慢、得之不易，故更顯其彌足珍貴。樟芝在台灣傳統醫藥歷史中始終被視為珍貴藥材，具有神奇的醫療保健功效。根據最近研究顯示，以2 % (v/v) 固態發酵樟芝乙醇萃取液處理人類肺癌A549細胞株72 小時，對A549細胞具有抑殺的效果。
在本實驗中，使用蛋白質體學技術來研究經固態發酵樟芝乙醇萃取液處理8小時的A549細胞和未經處理的A549細胞中表現量不同的蛋白質，利用PDQUEST軟體比對經處理及未經處理A549細胞的二維電泳圖譜，以找出表現差異2倍或2倍以上的蛋白質，在本論文中我們找到5個顯示統計上2倍或2倍以上顯著表現差異的蛋白質點（P<0.05）。
利用電灑游離式串連質譜儀鑑定這些表現差異蛋白質點，這五個蛋白質點分別被鑑定為 human galectin-1、human eukaryotic translation initiation factor 5A (eIf-5A)、Rho GDP dissociation inhibitor (Rho-GDI)、human calcium-dependent protease (CANP) and annexin V. 本論文在經固態發酵樟芝乙醇萃取液處理的A549細胞中找到五個表現差異的蛋白質，這些表現差異的蛋白質可以來幫助推測固態發酵樟芝乙醇萃取液如何影響腫瘤細胞的基因表達，以利未來進一步研究樟芝抑癌的機制。

Abstract
Antrodia camphorata (Chinese name, niu-chang-chih or niu-chang-ku) is a very rare and expensive mushroom because it slowly grows on the inner heart wood wall of the Cinnamomum kanehiraae only, which is an unique tree in Taiwan with limited amount. A. camphorata still has not been cultivated successfully. The potential health improvement and disease prevention of Antrodia camphorata have been emphasized in traditional Taiwanese herb medicine. Recent studies demonstrated 2 % (v/v) ethanol-extracts of A. camphorata cultivated by solid-state fermentation could effectively inhibit the proliferation of human lung carcinoma A549 cells in 72 hours.
In this study, we reported a proteomic experiment and showed the appreciable changes in protein synthesis after 8 hours treatment with 0.4% (v/v) ethanol-extracts of A. camphorata on A549 cells. After we compared two-dimension gel maps of non-treated with treated A549 cells using PDQUEST software, we have identified five protein spots,which were 2-fold or above differentially expressed with statistically significance（P<0.05）.
These five significantly downregulated protein spots were further identificatied by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI/MS/MS). These five proteins were identified as human galectin-1、human eukaryotic translation initiation factor 5A (eIf-5A)、Rho GDP dissociation inhibitor (Rho-GDI)、human calcium-dependent protease (CANP) and annexin V respectively. The results of this study help us to get insight into the effect of ethanol-extracts of A. camphorata cultivated by solid-state fermentation on the gene expression in the tumor A549 cells.

目 次
英文摘要 iv
中文摘要 v
誌謝 vi
目次 vii
表目錄 ix
圖目錄 x
符號與縮寫 xii
第一章 前言 1
1-1 背景的介紹 1
1-2 研究目的 3
第二章 文獻整理 4
2.1 樟芝的介紹 4
2-1-1 樟芝的特性 4
2-1-2 分類學 5
2-1-3 樟芝生理活性的研究 6
2-1-4 樟芝之藥理作用 7
2.2 蛋白質體學 9
2-2-1 蛋白質二維電泳 10
2-2-2 蛋白質鑑定技術 11
第三章 研究材料與方法 14
3-1 材料與藥品. 14
3-1-1 樟芝固態發酵乙醇萃取液 14
3-1-2 細胞株 14
3-1-3 藥品 14
3-1-3 實驗儀器 16
3-2 實驗方法 17
3-2-1 固體發酵樟芝乙醇萃取液對A549 細胞之處理 17
3-2-2 細胞蛋白質萃取液之製備 18
3-2-3 蛋白質液定量 18
3-2-4 蛋白質等電點聚焦電泳法 (isoelectric focusing, IEF) 18
3-2-5 SDS-聚丙烯膠電泳(SDS-polyacrylamide gel electrophoresis) 19
3-2-5 銀染 19
3-2-6 軟體比對 20
3-2-7 膠內蛋白質水解 20
3-2-7 電灑游離式串連質譜儀鑑定-Electrospray ionization tandem mass spectrometry (ESI/MS/MS) 20
3-2-8西方墨點法 21
第四章 結果 22
4-1 0.4 % (v/v)的固體發酵樟芝乙醇萃取液處理A549細胞存活測試 22
4-2 二維電泳圖譜條件之建立 22
4-3 以二維電泳圖譜分析固體發酵樟芝乙醇萃取液對人類A549肺癌細胞蛋白質圖譜的影響 23
4-4 受固體發酵樟芝乙醇萃取液影響而表現差異的蛋白質點 24
4-5 表現差異蛋白質點身份之確認 25
4-6以西方墨點法確認蛋白質表現的差異 25
第五章 討論 27
第六章 參考文獻 32
表目錄
表1 菇類的藥效成分 40
表 2. 未處理的人類肺癌細胞株（A549），經與固態樟芝發酵後的乙醇萃取液處理的人類肺癌細胞株細胞株中具有顯著性差異的蛋白質點 41
表3. 有差異之蛋白質點經LC/MS/MS所鑑定出的蛋白質資料 42
表4 、LC/MS/MS軟體與蛋白質資料庫比對的相關參數定義 43
圖目錄
圖2-1 樟芝（Antrodia camphorata） 44
圖2-2 樟芝子實體萃取物中發現新的三帖類化合物 45
圖2-3 樟芝子實體萃取物中發現新的三帖類化合物 46
圖2-4 由樟芝分離得的十二種化合物 47
圖2-6 固態樟芝發酵後的熱水及乙醇萃取物作用於 A549 細胞的實驗結果 48
圖2-7 固態樟芝發酵後的熱水及乙醇萃取物作用於 Hep3B 細胞的實驗結果 48
圖2-8 蛋白質體學簡化流程圖 49
圖4-1 0.4 % (v/v)的固態發酵樟芝乙醇萃取液處理0、2、4、6、8小時以及0.4% (v/v)固體培養基基材乙醇萃取液與100%乙醇（固體發酵樟芝是經乙醇萃取）處理 A549細胞8小時的細胞存活率 50
圖4-2. E7 cells 細胞二維電泳圖譜 51
圖4-3 二維電泳條件改進後之E7 細胞二維電泳圖譜 52
圖4-4 （A）為未處理的人類肺癌細胞株（A549） 53
圖4-4 （B）固態樟芝發酵後的乙醇萃取液處理的人類肺癌細胞株（A549） 8小時的代表性二維電泳圖譜 54
圖4-5 （A）未處理的人類肺癌細胞株中蛋白質點的二維電泳圖譜放大圖（B）同區域的固態樟芝發酵後的乙醇萃取液處理的人類肺癌細胞株的二維電泳圖譜放大圖 56
圖4-6 未處理的人類肺癌細胞株（A549），經與固態樟芝發酵後的乙醇萃取液處理的人類肺癌細胞株細胞株中具有顯著性差異的蛋白質點的平均volume數值 57
圖.4-7 （A）Spot 1：Human Galectin-1蛋白質點LC/MS/MS比對的結果。 58
圖.4-7 （B）Spot 2：Human translation initiation factor 5A蛋白質點LC/MS/MS比對的結果 59
圖.4-7 （C）Spot 3：Rho GDP dissociation inhibitor蛋白質點LC/MS/MS比對的結果 60
圖.4-7 （D）Spot 4：Human Calcium-dependent proteas 蛋白質點LC/MS/MS比對的結果 61
圖.4-7 （E）Spot 5：Human Annexin A5蛋白質點LC/MS/MS比對的結果。 62
圖4-8 (A)∼(D)有差異蛋白質點之西方墨點法的結果。 66

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