臺灣博碩士論文加值系統

樟芝是一種用來抗癌和抗發炎的台灣民間著名醫藥蕈類 (無褶菌目，多孔菌科)。然而，目前對於不同樟芝萃取物影響白血病細胞生長抑制的分子機制尚未明瞭。在本研究中，我們結合草藥鑑定、UV/VIS光譜法、高效液相層析法與感應偶合電漿質譜法建構一個整合性高效率平台，來將草藥藥材進行標準化。同時，我們以cDNA微陣列科技來檢視不同樟芝萃取物(水萃與醇萃物)對KG-1(急性骨髓性白血病細胞株)細胞的生長抑制影響。基於此科學平台，我們發展出一個可行的定量方法並建立出有機/無機的化學指紋。透過比較草藥萃取物對KG-1整體基因的表現型式來看，數個可能的生物反應與相關的標的基因，將可被以基因本體論(Gene Ontology)為基礎的群集方法所鑑定出來。從兩種萃取物的基因本體論歸類比較之顯著性來看，結果暗示樟芝水萃物會下調分解與碳水化合物代謝相關的基因群，而樟芝醇萃物則會促進MHC class II 抗原呈現相關基因群的表現。

Antrodia camphorata ( A. camphorata ) is well known in Taiwan as a folk medicinal fungus with anticancer and anti-inflammatory effects. However, the molecular mechanism underlying leukemia growth inhibition effects through different A. camphorata treatment is poorly understood. In this study, we constructed an integrated high-throughput platform to standardize the herbal materials by using a combination of herbal identification, UV-VIS spectrophotometry, high performance liquid chromatography (HPLC), and inductively coupled plasma mass spectrometry (ICP-MS). Simultaneously, we used cDNA microarray technology to examine the effects of various A. camphorata mycelia extracts (water and methanol extracts) on the effects of growth inhibition on KG-1 cells (human acute myelogenous leukemia cell line). Based on this science-based platform, we developed a practical quantitative method and established an organic/inorganic chemical fingerprint. By the comparison of KG-1 global gene expression patterns from herbal extracts, several possible biological responses and relevant marker genes were identified by gene ontology (GO)-based clustering analysis. The statistically significantly comparison of GO category from both treatments indicated that water extract could down-regulate carbohydrate catabolism related genes and methanol extract up-regulate MHC class II antigen processing genes, respectively.

目 錄
中文摘要 2
英文摘要 3
致謝 4
第一章 序論
1.1 前言 13
1.2 研究動機 14
1.3 研究目的 14
1.4 研究方法 15
第二章 文獻考察
2.1 樟芝背景資料 16
A. 樟芝歷史淵源 16
B. 樟芝的命名、特徵與分佈 17
C. 比較靈芝與樟芝的有效成分、萃取方式 18
D. 樟芝的療效 20
2.2 KG-1細胞株背景資料 20
2.3 草藥整合性高效率平台建立與cDNA微陣列系統 20
A. 草藥相關的基因體研究 20
B. cDNA微陣列系統回顧 21
第三章 原理方法與材料設備
3.1 草藥來源與成分分析 23
A. 草藥來源、保存與製備 23
B. 樟芝的鑑定 23
C. 樟芝有效成分萃取方式 23
D. 光譜分析 24
E. 高效液相層析法 24
F. 微量金屬分析 24
3.2 樟芝萃取液對急性骨髓性白血病細胞株影響 25
A. 細胞株來源、保存與培養 25
B. 細胞生長曲線 25
C. 細胞存活率分析 25
3.3 微陣列基因晶片實驗系統調整、流程與驗證 26
A. 點印系統最優化 26
B. 探針點印 26
C. 晶片處理 27
D. 檢體製備 28
E. 反轉錄與螢光標定 29
F. 雜合反應與清洗 29
G. 影像掃瞄與篩選 30
H. 資料分析 30
第四章 實驗設計與結果討論
4.1 實驗設計 35
A. 草藥的定性定量 35
B. 細胞培養控制與草藥療效評估 35
C. 微陣列實驗系統誤差矯正 35
4.2 樟芝整合性高效率平台與cDNA微陣列篩選系統 38
A. 樟芝菌絲體鑑定結果 38
B. 水、醇萃取物結果 38
C. 光譜分析結果 38
D. 高效液相層析法結果 42
E. 感應偶合電漿質譜分析 42
F. 細胞生長曲線與存活率分析 43
G. 比較兩種樟芝萃取物對細胞生長抑制影響 44
H. 微陣列基因晶片實驗設計與結果 44
第五章 結論 51
第二部分 微陣列基因晶片系統實驗：綠螢光信號系統偏差之探究
1.1 研究背景 53
1.2 實驗設計 53
A. 螢光光譜量測 53
B. DNA微陣列實驗 54
1.3 結果討論 55
A. 螢光光譜測量結果 55
B. DNA微陣列實驗結果 55
C. 點印大小與濃度影響 56
參考文獻 58







圖 表 目 錄
圖一. 樟芝菌絲體 63
圖二. 三萜類化合物 63
圖三. 三萜類化合物 64
圖四. 固醇類、三萜類化合物 65
圖五. 固醇類化合物 66
圖六. 木脂素與甲基酯 67
圖七. 倍半萜內酯 68
圖八 苯環化合物 69
圖九. 順丁烯二酸、丁二酸 70
圖十. cDNA microarray 的原理流程圖 71
圖十一. 樟芝種源鑑定區域示意圖 71
圖十二. 樟芝序列比對結果 72
圖十三. 將樟芝直接以水萃取，並稀釋二十倍所得光譜圖 73
圖十四. 個別將光譜圖疊合後所得之光譜分布 73
圖十五. 水萃物個別以系列稀釋後所得光譜圖、及針對特定三波長之吸收值迴歸趨勢評估 74
圖十六. 醇萃物個別以系列稀釋後所得光譜圖、及針對特定三波長之吸收值迴歸趨勢評估 75
圖十七. 醇萃物依比例稀釋光譜圖 76
圖十八. 樟芝萃取物濃縮前後稀釋光譜比較圖 77
圖十九. 樟芝樟芝水萃物在257 nm波段所得系列稀釋圖譜、樟芝樟芝醇萃物在228 nm波段所得圖譜 78
圖二十. 微量金屬含量圖 79
圖二十一. 細胞生長曲線圖 79
圖二十二. 樟芝水萃物、醇萃物對KG-1絕對細胞生長抑制圖 80
圖二十三. 樟芝萃取物抑制65%KG-1細胞生長之濃度(IC65) 82
圖二十四. 毛細管電泳圖、及進行微陣列實驗前樣本的RNA品質評估圖 83
圖二十五. 利用Visual C++運算得到有效(efficiency=0.9)迴圈設計圖 84
圖二十六. 將紅綠光訊號散佈圖轉換為MA圖、歸一化後MA圖和條件篩選後的MA圖 85
圖二十七. 添加的十群控制組基因螢光訊號分佈長條圖 86
圖二十八. 為九片微陣列晶片篩選後的MA圖和添加控制組的訊號分布長條圖 86
圖二十九. 3956個基因之表現量差異分佈圖 89
圖三十. 以群集方法評估微陣列系統實驗對基因表現的影響 90
圖三十一. 以基因本體論為基礎的群集分析法圖 91
圖三十二. 以基因本體論為基礎的K-means群集法圖 92
圖三十三. 標的基因分別與3956個可信基因進行本體論註解數目上的比較圖 93
圖三十四. 以PathwayAssist軟體所描繪出的分子信息網絡圖 93
圖三十五. 樟芝水萃物所影響的生化反應路徑 94
圖三十六. ＤＮＡ微陣列點印設計圖 95
圖三十七. 綠螢光染劑稀釋一千倍之光譜圖 95
圖三十八. 顯示二次水中不含任何螢光物質 96
圖三十九. 95%乙醇和異丙醇在630 nm時有極微弱背景值 96
圖四十. 一般常用於點印的點印液 97
圖四十一. 目前用於本實驗室微陣列點印系統的3X SSC點印溶液 97
圖四十二. DNA加入3X SSC點印溶液 97
圖四十三. 處理晶片後三階段時的影像檔 98
圖四十四. 兩重複各三階段晶片的實驗螢光強度長條圖、箱型圖 99
圖四十五. 比較兩重複晶片實驗在雜合反應清洗後各區塊螢光值 100
圖四十六. 兩重複晶片實驗雜合反應清洗後區塊間相關性分析圖 100
圖四十七. 含點內螢光值區塊內的點大小分布圖 101
圖四十八. 兩重複實驗中各區塊的點內螢光值與點大小之關係圖 101
圖四十九 以同一種點印液(3XSSC)點印進行另外兩重複實驗的螢光值分佈圖 102
圖五十 為48根針所造成的區塊分布圖 103

表一. 萃取產率估計表 104
表二. 樟芝療效文獻整理表 105
表三. 微陣列系統誤差因素表 107
表四. 樟芝萃取產量估計表 108
表五. 水萃物相對定量表 108
表六. 醇萃物相對定量表 108
表七. 醇萃物溶解情形定量表 108
表八 水萃物指標性成分定量表 108
表九 醇萃物指標性成分定量表 109
表十 水、醇萃物所造成基因表現對立假設 109
表十一 水萃標的基因表 110
表十二 醇萃標的基因表 114

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