臺灣博碩士論文加值系統

全球約有 20 %的人口受到過敏性疾病的困擾，其中第I型過敏反應的症狀， 包括過敏性皮膚炎，過敏性鼻炎，過敏性哮喘，過敏反應和食物過敏。蛹蟲草是一 種傳統中藥材，擁有許多治療功效，例如抗腫瘤，抗氧化和免疫調節...等。在本研 究中，我們利用蛋白質體學技術來尋找家蠶蛹蟲培養之蛹蟲草子實體乙醇萃取物 (CM_EE)對 rat basophilic leukemia cells (RBL-2H3)蛋白質表現影響，並進一步發現 由 IgE 所引發過敏反應的分子機制，我們利用二維膠體電泳(2-DE)找尋有加入與未 加入 CM_EE 的 RBL-2H3 細胞蛋白質表現差異，經蛋白質體圖譜相互比對發現有 13 個蛋白質點具有 2 倍差異，其中 10 個點為表現量下降，3 個點為表現量上升， 由 Ingenuity Pathways Analysis (IPA)分析顯示，這些蛋白質主要功能分別屬於 Transcription Regulator、Enzyme、peptidase、Translation Regulator。

About 20% of the global population is affected by allergy, type 1 hypersensitivity, including atopic dermatitis, allergic rhinitis, allergic asthma, anaphylaxis, and food allergy. Cordyceps militaris is a popular traditional Chinese medicinal fungus. Cordyceps militaris has numerous therapeutic values, such as anti-tumor, anti-oxidation and immunomodulatory effects. The proteomics method to search for the ethanol extract of Cordyceps militaris fruit body (CM_EE)-affected genes that might clarify the molecular mechanism underlying IgE-mediated allergic response. We underwent two-dimensional gel electrophoreses (2-DE) to acquire the proteomic profiles of CM_EE-treated and naive rat basophilic leukemia cells (RBL-2H3). The proteome maps of treated basophilic leukemia cell were compared with those of un-treated cell to find the differentially express proteins. Comparative proteomics indicated that 13 dys-regulated proteins were present in CM_EE-incubated RBL-2H3 cells with 10 down-regulated proteins and 3 up- regulated proteins. Ingenuity Pathways Analysis (IPA) analysis indicate that the main functions of these proteins belong to Transcription Regulator, Enzyme, peptidase, and Translation Regulator respectively.

中文摘要 ············································································ II
ABSTRACT ········································································· III
誌謝 ··············································································· IV
目錄 ··············································································· VI
表目錄 ············································································ VIII
圖目錄 ············································································· IX
附表目錄 ··········································································· X
附圖目錄 ··········································································· XI
縮寫對照表 ········································································ XII
第一章 前言··········································································1
第二章 文獻回顧······································································2
2.1 蛹蟲草的介紹······································································2
2.2 蛹蟲草與冬蟲夏草的比較···························································3
2.3 蛹蟲草的藥理活性································································4
2.4 免疫過度敏感反(HYPERSENSITIVITY)···············································5
2.5 免疫過度敏感反應之類型···························································5
2.5.1 第I型免疫過度敏感反應 - IgE 誘導的過敏反應 ·······································5
2.5.2 第II型免疫過度敏感反應 - 抗體媒介細胞毒殺型過敏反應 ······························5
2.5.3 第III型免疫過度敏感反應 - 免疫複合體媒介型過敏反應 ·······························6
2.5.4 第IV型免疫過度敏感反應 - 細胞媒介型過敏反應 ·····································7
2.6 過敏的治療方式···································································7
2.6.1 藥物治療········································································8
2.6.2 減敏治療········································································8
2.7 IGE 抗體引發之訊息傳遞 ···························································8
2.8 蛋白質體學······································································ 10
2.9 蛋白質二維電泳·································································· 11
2.10 蛋白質鑑定技術 ······························································· 12
第三章 研究材料與方法 ······························································ 14
3.1材料與藥品······································································· 14
3.1.1 蛹蟲草(Cordyceps militaris,CM)·················································· 14
3.1.2 細胞株········································································14
3.1.3 藥品試劑·····································································14
3.1.4 實驗儀器······································································17
3.2 研究方法···································································· 19
3.2.1 蛹蟲草乙醇萃取物之製備 ······················································· 19
3.2.2 RBL-2H3 細胞蛋白質萃取液之製備 ··········································· 19
3.2.3 蛋白質液定量·······························································20
3.2.4 蛋白質等電點聚焦電泳法(Isoelectric focusing, IEF)························ 20
3.2.5 膠條平衡(Equilibration)··························································21
3.2.6 SDS -聚丙烯膠電泳(SDS-polyacrylamide gel electrophoresis) ············· 21
3.2.7 銀離子染色(Silver staining) ····················································· 21
3.2.8 二維電泳膠片影像分析比對····················································22
3.2.9 統計軟體·····································································22
3.2.10 蛋白質身分鑑定(ESI-tandem/MS/MS)·······································22
3.2.11 西方墨點法(Western blotting) ················································· 24
第四章 結果·························································26
4.1 家蠶蛹蟲培養之蛹蟲草子實體乙醇萃取物(CM_EE)對 RBL-2H3 細胞二維電 泳圖譜條件的建立·········································································· 26
4.2 尋找表現差異的蛋白質點····················································26
4.3 鑑定蛋白質的身份···························································· 27
4.4 驗證二維電泳之蛋白質表現量 ················································· 28
4.5 CM_EE 對 RBL-2H3 細胞之 BIP 蛋白質表現量影響 ···························· 29
第五章 討論·····································································30
第六章 參考文獻 ··································································· 35

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