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研究生:陳美如
研究生(外文):Mei-Ju Chen
論文名稱:天麻免疫調節蛋白之純化與活性分析
論文名稱(外文):Purification and Function Analysis of the Immunomodulatory Protein from Gastrodia elata
指導教授:許輔許輔引用關係
口試委員:周志輝潘敏雄
口試日期:2015-07-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:66
中文關鍵詞:天麻純化免疫調節蛋白
外文關鍵詞:Gastrodia elataPurificationImmunomodulatory Protein
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Tianma (Gastrodia elata), also known as fixed wind grass or barbed arrows, is a medicinal orchid used in traditional Chinese medicine to treat vertigo, paralysis, convulsions, cramps, epilepsy, high blood pressure and other symptoms. We have previously purified an immunomodulatory protein IPAF from Anoectochilus formosanus. The protein IPAF has an amino acid sequence highly similar to proteins from Orchidaceae plants, including Epipactis helleborine lectin, Listera ovata lectin, and G. elata-gastrodianin-4B. Therefore, we hypothesized that Orchidaceae plants might possess proteins similar to IPAF. To test our hypothesis, the crude protein was extracted from G. elata, Dendrobium huoshanense, Dendrobium nobile, and Dendrobium loddigesii and then analyzed by polyacrylamide gel electrophoresis and Western blotting. Subsequently, a 15.1 kDa protein G. elata (GEP), which exhibited similar biochemical characteristics to IPAF, was purified via anion exchange chromatography using DE-52 and HiTrap Q columns. Finally, RAW 264.7 murine macrophage cells were used for preliminary activity tests. GEP (5 μg/mL) could activate nitric oxide (NO) (10.4 ± 0.6 μM), TNF-α (935.4 ± 118.5 pg/mL), and IL-1β (25.1 ± 3.0 pg/mL) secretion by RAW 264.7 murine macrophage. In addition, GEP enhanced nucleic acid replication by 1.16-fold as analyzed by BrdU analysis, and promoted 1.43-fold of MTT metabolic activity in murine splenocytes. In this study we purified a new orchid protein, which exhibited similar biochemical characteristics as the IPAF, and we also assessed its immunomodulatory function. GEP activated RAW 264.7 macrophages by enhancing NO, TNF-α and IL-1β secretion. Moreover, GEP also stimulated cell proliferation in mouse splenocytes. On the basis of previous the findings, in our laboratory GEP, as well as other orchid proteins, might play important roles in the medicinal functions of Orchidaceae plants.

誌謝 I
摘要 III
Abstract IV
目錄 VI
圖目錄 IX
Chapter 1 緒論 1
1.1 序言 1
1.1.1 天麻典故 1
1.1.2 天麻之分部和栽培 1
1.1.3 天麻之傳統藥理與藥用潛力 3
1.1.4 天麻之生理活性 3
1.2 台灣金線連 8
1.3 免疫調節 10
1.3.1 免疫功能 10
1.3.2 發炎反應 11
1.3.3 巨噬細胞的免疫系統角色 12
1.4 研究動機與架構 16
Chapter 2 材料與方法 17
2.1 天麻、霍山石斛、川石斛和金釵石斛等四種蘭科植物粗蛋白製備 17
2.1.1 天麻、霍山石斛、川石斛和金釵石斛等四種蘭科植物粗蛋白萃取 17
2.1.2 天麻、霍山石斛、川石斛和金釵石斛粗蛋白之特性分析 19
2.2 天麻蛋白 GEP 純化 23
2.2.1 DE-52 離子交換樹脂層析 23
2.2.2 快速蛋白質層析系統(FPLC SYSTEM)分析 25
2.3 天麻蛋白 GEP 免疫調節活性試驗 25
2.3.1 一氧化氮 (NITRIC OXIDE) 產量分析 25
2.3.2 Α腫瘤壞死因子 (TNF-α) 產量之分析 27
2.3.3 介白質 (IL-1β) 產量之分析 28
2.3.4 小鼠脾細胞之取得 29
2.3.5 脾臟細胞的代謝活性 (MTT ASSAY) 29
2.3.6 脾臟細胞的細胞增生效應 (BRDU ASSAY) 30
2.3.7 Γ干擾素 (IFN-γ)產量之分析 31
Chapter 3 結果 32
3.1 四種藥用蘭科植物全蛋白萃取及特性分析 32
3.1.1 聚丙烯醯胺膠體電泳分析 32
3.1.2 西方轉漬分析 32
3.2 天麻蛋白 GEP 的純化 33
3.2.1 DE-52陰離子交換樹脂 33
3.2.2 快速蛋白層析系統 (FPLC SYSTEM) 分析 33
3.3 天麻蛋白 GEP 的免疫調節活性 34
3.3.1 天麻蛋白 GEP 對 RAW 264.7 MACROPHAGES 之影響 34
3.3.2 天麻蛋白 GEP 對小鼠脾細胞之影響 38
Chapter 4 討論 42
Chapter 5 結論 45
參考文獻 47
圖表 55


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