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研究生:黃泓誠
研究生(外文):HUANG HONG-CHENG
論文名稱:以蛋白質體學技術探討酒精熱萃取樟芝液對人類非小細胞A549細胞株的蛋白質差異表現
論文名稱(外文):Proteomic search for the target genes of ethanol extract from Antrodia Camphoratamycelia against human non-small cell lung carcinoma A549 cell
指導教授:吳定峰吳定峰引用關係
指導教授(外文):WU TING-FE
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:61
中文關鍵詞:樟芝
外文關鍵詞:antrodia camphorata
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牛樟芝在台灣是非常有名的傳統中草藥,並且它已經被發現具有抗氧化、抗發炎、以及抑制癌症的活性。在實驗室先前的研究中,牛樟芝菌絲體中的化合物組成以極性來分成幾部份。菌絲體使用了丙酮、乙酸乙酯、酒精還有水來萃取。在MTT測試中顯現出酒精熱萃取物對人類A549細胞株有極強的抑制活性。本篇實驗中,大量的細胞凋亡研究論證了酒精熱萃取樟芝誘導了人類A549細胞株經由觸發內質網和粒線體路徑進行凋亡。此外,A549細胞加入樟芝及未加入所呈現的三對二維電泳膠圖,以PDQUEST軟體分析後,發現了19個具有二倍以上差異且P值小於0.05和出現機率大於六分之三的蛋白質點。這19個具有顯著性差異的蛋白質被挑選出以液相層析電噴灑串聯式質譜儀最為稍後的驗證。經鑑定後找出了8個相關表現量上升的蛋白質點,分別為N-acylaminoacyl-peptide hydrolase, 60 kDa heat shock protein, PAK 2, aldehyde dehydrogenase 2, proteasome 26S subunit, actin related protein 2/3 complex, actin alpha 2 。另9個表現量下降的蛋白質點分別是PS1TP5BP1 , PRSS3 protein, Phosphatase 2a, tubulin alpha, eIF4A1, NDUFS1, T-complex protein 1 subunit beta, ATPase, H+ transporting, lysosomal 56/58kDa 。
Antrodia camphorata is well known in Taiwan as traditional Chinese medicine, and it has been shown to exhibit antioxidant, anti-inflammatory and anticancer effects. In our previous study, the compounds present in A. camphorata mycelia were fractionated dependent upon the polarity. The mycelia were extracted with the order of hexane, ethylacetate, ethanol and water respectively. MTT assay showed that the ethanol fraction (EC) has the most profound inhibitory effects on human A549 cells. In this study, extensive apoptotic investigations demonstrated that EC could induce human A549 cell apoptosis via triggering ER stress and the mitochondrial pathway. Furthermore, two-dimensional (2-D) gel maps of non-treated and treated human A549 cells were compared using PDQUEST analytical software to discover 19 statistically significant 2-fold or above-2-fold differentially-expressed protein spots (p<0.05, student’s t test) as observed in at least 3out of 6 comparisons. The 19 protein spots that were significantly de-regulated were chosen for subsequent identification by high performance liquid chromatography electro-spray tandem mass spectrometry. Of these up-regulated proteins, N-acylaminoacyl-peptide hydrolase, 60 kDa heat shock protein, PAK 2, aldehyde dehydrogenase 2, proteasome 26S subunit, actin related protein 2/3 complex, actin alpha 2, and down-regulated proteins, PS1TP5BP1 , PRSS3 protein, Phosphatase 2a, tubulin alpha, eIF4A1, NDUFS1, T-complex protein 1 subunit beta, ATPase, H+ transporting, lysosomal 56/58kDa were associated with the carcinogenesis.
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中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
縮寫對照表 viii
第一章 前言 1
第二章 文獻探討 2
2-1. 牛樟芝的介紹 2
2-2. 牛樟芝的特性 2
2-3. 牛樟芝的分類學 2
2-4. 牛樟芝的成分與藥理活性 3
2-4-1. 牛樟芝的保肝作用 3
2-4-2. 牛樟芝的抗發炎作用 3
2-4-3. 牛樟芝的提升免疫能力作用 4
2-4-4. 牛樟芝的抗氧化作用 4
2-4-5. 牛樟芝的抑制腫瘤生長 4
2-5. 蛋白質體學 5
2-6. LAVAPurple 6
第三章 材料與方法 7
3-1. 材料與藥品 7
3-1-1.細胞株 7
3-1-2.樟芝 7
3-1-3.藥品試劑 7
3-1-4.實驗儀器 10
3-2. 實驗方法 11
3-2-1. 牛樟芝固態發酵萃取液之製備 11
3-2-2. Human A549 cell 蛋白質萃取液之製備 11
3-2-3. 蛋白質液定量 11
3-2-4. 蛋白質等電點聚焦電泳法(isoelectric focusing,IEF) 12
3-2-5. 膠條平衡(Equilibration) 12
3-2-6. SDS-聚丙烯膠電泳(SDS-polyacrylamide gel electrophoresis) 13
3-2-7. LavaPurple螢光染色 13
3-2-8. 軟體比對 13
3-2-9. 統計軟體 14
3-2-10. 蛋白質鑑定 14
3-2-11. Western Blotting 15
第四章結果 16
4-1. 研究目的 16
4-2. 人類非小細胞A549 cell二維電泳圖譜條件的建立 16
4-3. 人類非小細胞A549 cell加樟芝與未加樟芝的蛋白質圖譜 18
4-4. 尋找表現差異的蛋白質點 18
4-5. 鑑定蛋白質的身分 19
4-5. Western Blotting 20
第五章 討論 21
第六章 參考文獻 25
1. Wu, S.H., Ryvarden, L., Chang, T.T., Antrodia camphorata (―niu-chang-chih‖),new combination of a medicinal fungus in Taiwan. Botanical Bulletin of Academia Sinica 1997,38, 273–275.
2. Zang, M., Su, Q.H., Ganoderma comphoratum, a new taxon in genus Ganoderma from Taiwan, China. Acta Botanica Yunnanica 1990,12, 395–396.
3. Chang, C.Y., Lue, M.Y., Pan, T.M., Determination of adenosine, cordycepin and ergosterol contents in cultivated Antrodia camphorata by HPLC method. Journal of Food and Drug Analysis 2005,13, 338–342.
4. Zong, H. A., Zheng, X., Zhen, L., Hong, X., Xiao, Z.,Wen,F.D. Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases. Journal of Ethnopharmacology 2009,121, 194–212.
5. Lu ZM, Tao WY, Zou XL, Fu HZ, Ao ZH. Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats. Journal of Ethnopharmacology 2007,110(1): 160–164.
6. Liu DZ, Liang HJ, Chen CH, Su CH, Lee TH, Huang CT, Hou WC, Lin SY, Zhong WB, Lin PJ, Hung LF, Liang YC. Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action. J Ethnopharmacol. 2007 Aug 15;113(1):45-53.
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