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研究生:林惠君
研究生(外文):Huey-Jun Lin
論文名稱:薑黃素抗IgE媒介型過敏反應之體外效用評估
論文名稱(外文):In vitro assessment of curcumin on IgE-mediated allergic responses
指導教授:龔瑞林龔瑞林引用關係
指導教授(外文):Zwe-Lin Kong
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:120
中文關鍵詞:薑黃素IgE媒介型過敏反應脫顆粒活性氧化物PKC deltaFc epsilon RI
外文關鍵詞:CurcuminIgE-mediated allergic responsesdegranulationROSPKC deltaFc epsilon RI
相關次數:
  • 被引用被引用:10
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中文摘要

過敏反應為個體對環境抗原產生不恰當免疫反應所致,造成肥大細胞及嗜鹼性白血球等過度活化,常見之過敏性疾病包含氣喘、過敏性鼻炎及異位性皮膚炎等,皆屬於第一型過敏反應 (Type I allergic response),為IgE所主導之立即型過敏反應 (IgE-mediated allergic response )。文獻指出IgE媒介之過敏反應與胞內活性氧物質(reactive oxygen species; ROS) 之產生密切相關,ROS扮演著初級訊息傳遞調控者,調控著過敏反應相關訊息傳遞分子之表現。薑黃素為鬱金類植物地下根主要之黃色色素,為天然界豐富之多酚類成分,透過結構上methoxy group及b-diketone等可直接捕捉活性氧物質或間接抑制相關基因表現,目前已知其具有抗氧化、抗發炎及抗癌等重要生理活性。本論文進一步探討薑黃素之抗氧化特性在抗過敏反應之效用,以老鼠肥大細胞株RBL-2H3及人類嗜鹼性白血球細胞株KU812作為實驗對象,探討薑黃素對IgE所誘發之肥大細胞脫顆粒現象之緩解及過敏相關基因表現量。實驗於IgE致敏模式及A23187兩系統下進行,實驗證實IC50= 5 uM及IC50= 10 uM之薑黃素可分別抑制此兩系統所誘發之細胞脫顆粒介質釋出,隨薑黃素濃度之增加,其對胞內ROS之生成亦有顯著抑制效果。ROS可調節過敏相關訊息分子之活化與位移,薑黃素透過對ROS生成之抑制而影響PKC-�啎圻麮鴃A由本實驗結果可推論薑黃素抑制細胞脫顆粒現象可能與此機制相關。FceRI為IgE之接受體,由abr2四個胜肽所組成,過敏反應中此接受體呈高度活化狀態,本論文證實KU812細胞於50 uM薑黃素處理12或24小時,其IgE主要結合區域FceRI表現量分別減少20%及50%,而FceRI表現量於24小時10uM薑黃素處理下減少40%。另,KU812細胞所分泌Th2相關激素IL-4及IL-13,於10uM薑黃素處理30分鐘其相關基因表現量亦減少80%。由本論文實驗結果推論,薑黃素可有效緩解IgE媒介之第一型過敏反應,且其作用機制可能與FceRI及Th2細胞激素等過敏相關基因表現量顯著相關。

關鍵字: 薑黃素、IgE媒介型過敏反應、脫顆粒、活性氧化物、PKC-gamma、FceRI
Abstract

Allergy owing to inappropriate immune responses to antigen in the environment. Asthma, allergic rhinitis and atopic dermatitis are common diseases which all belong to Type I allergic responses (so called IgE-mediated allergic responses). In recent study, it shows higher relationship between ROS and allergic responses. Curcumin is a major yellow pigment isolated from the spice turmeric. Dietary curcumin possesses an anti-oxidant and anti-inflammatory properties and it also exhibits significant chemopreventive activity. Rat basophilic leukemia cells (RBL -2H3) express α, β and γ chain of Fc�悐I, which plays a major role in the IgE-mediated allergic response. Antigen-IgE crosslinking initiates the release of ��-hexosaminidase, which is a marker of degranulation in RBL-2H3. Curcumin shows activity against ��- hexosaminidase release from IgE-mediated at 5 �嵱, even effective in calcium ionophores (A23187)-induced degranulation at��10 �嵱. Furthermore, effects of curcumin on antigen/A23187 induced release of histamine were also examined, both of them were decreased accompanied with the increasing concentration of curcumin. Among IgE-mediated allergic responses, antigen induces mast cells intracellular ROS production significantly. Curcumin could reduce ROS production from IgE-mediated or A23187 induced at 5 �嵱, and inhibits PKC-�� translocation from cytosolic to particulate.
Fc�悐I consists of four subunits (������2), and the �� chain is high affinity to IgE. The surface expression of Fc�悐I in human basophilic cell line (KU812) also studied, treating with dose-dependent curcumin expresses lower level of mRNA expression of Fc�悐I ���nand ���nchain. Even 10 �嵱 curcumin could also suppress IL-4/IL-13 mRNA expression. And all above, these results suggest that curcumin can alleviate the IgE-mediate allergic responses through down-regulating Fc�悐I expression and reducing the production of the allergic mediates and ROS.

Key words: Curcumin, IgE-mediated allergic responses, degranulation, ROS, PKC-���z Fc�悐I
目錄

謝辭 ……………………………………………………………………i
名詞縮寫………………………………………………………………iii
中文摘要 ………………………………………………………………v
英文摘要 ……………………………………………………………vii
壹、緒論 ………………………………………………………………1
貳、文獻回顧 …………………………………………………………5
ㄧ、過敏簡介 ……………………………………………………. 5
(一) 過敏 …………………………………………………….5
(二) 過敏疾病致病機轉 ………………………………….....6
1. 早期反應 ……………………………………………..6
1-1. 過敏原之辨識 ……………………………………..6
1-2. 輔助性T細胞之活化 ……………………………. 7
1-3. B細胞的活化及IgE抗體之生成 ………………….7
1-4. 致敏化之肥大細胞及嗜鹼性白血球………………8
2. 晚期反應 …………………………..…………………8
(三) 抗過敏藥物與其他抗過敏物質………………………….9
1. 皮質類固醇(Corticosteroids)……………………….9
2. 介質拮抗劑 (Mediator antagonists) ………………10
3. 細胞激素調節劑(Cytokine modulators) ……………11
4. 專一抗過敏藥物(Specific anti-allergic drugs)…13
5. 其他已知抗過敏成分 …………………………………14
(四) 過敏反應相關之訊息傳遞…………………….………14
1. FceRI ………………………………………………….14
2. ROS …………………………………………………….15
3. 鈣離子…………………………………………………17
4. PLC-g�n…………………………………………………18
5. PKC-�唌K………………………………………………18
二、薑黃素 …………………………………………………… 19
(一) 薑黃素簡介 ………………………………………… 19
(二) 薑黃素之生化代謝 ………………………………… 20
(三) 薑黃素之生理機能活性 …………………………… 20
1. 抗腫瘤 …………………………………………….. 20
2. 抗氧化 …………………………………………….. 21
3. 抗發炎 …………………………………………….. 21
4. 抗過敏 …………………………………………….. 22
參、整體實驗設計…………………………………………………….23
肆、實驗材料與方法 …………………………………………………24
一、 細胞株與細胞培養 ……………………………………....24
二、 藥品與試劑 …………………………………………………24
三、 實驗方法 ……………………………………………………27
1. 細胞存活率試驗(MTT assay) ……………………...27
�n�n�n�n�n2. b-hexosaminidase 釋放測定 ……27
3. Lactate dehydrogenase (LDH)活性評估 …………29
4. 組織胺(Histamine)釋放測定 ………………………29
5. 胞內活性氧分析 …………………………………...30
6. 過氧化氫酶活性測定 ……………………………...31
7. 西方轉漬法分析訊息傳遞蛋白PKC�{���n�|�|�|�|�|�|
8. RT-PCR分析KU812 FceRI 表現 ………………….33
9. 西方轉漬法分析FceRI表現量 ……………………34
10. RT-PCR分析KU812 Th2 cytokines表現 …………35
四、統計方法……………………………………………………36
伍、結果與討論………………………………………………………37
一、薑黃素對細胞存活率之影響 ……………………….....37
二、建立抗原實驗模式探討RBL-2H3細胞之脫顆粒作用…….37
三、建立Ca2+ ionophore A23187 實驗模式探討RBL-2H3細胞
之脫顆粒作用 ………………39
四、薑黃素對RBL-2H3細胞脫顆粒作用之影響 ……………..39
五、薑黃素對RBL-2H3細胞組織胺釋放之影響 ……………..42
六、薑黃素對RBL-2H3細胞內ROS生成之抑制 ……………43
七、薑黃素對RBL-2H3細胞內CAT活性之影響 ……………45
八、薑黃素對訊息分子PKC-�啈麮黎尬v響 ……………………46
九、薑黃素對KU812細胞表面Fc�悐I表現量之影響 …………47
十、薑黃素對KU812 Th2 細胞激素表現量之影響 …………..49
陸、總結………………………………….……………………………51
柒、參考文獻 ………………………………………………………..53
捌、圖表 …………………………………..………………....……66
玖、附錄 ………………………………………………………………94





圖表目錄

圖一、整理IgE媒介型過敏反應機制圖………………………………66
圖二、肥大細胞與嗜鹼性白血球分泌Th2細胞激素影響B細胞IgE生成……………………………………………………………………67
圖三、薑黃素對RBL-2H3細胞存活率影響…………………………68
圖四、薑黃素對KU812細胞存活率之影響…………………………69
圖五、抗體抗原(anti-DNP IgE-/DNP-BSA)刺激脫顆粒反應最適濃度曲線……………………………………………………………………70
圖六、不同濃度A23187刺激RBL-2H3細胞之脫顆粒作用………71
圖七、組織胺標準曲線 ……………………………………………72
圖八、不同濃度A23187刺激RBL-2H3細胞組織胺釋放 …………73
圖九、薑黃素抑制IgE媒介之脫顆粒反應 …………………………74
圖十、薑黃素對細胞LDH釋放量之影響 ……………………………75
圖十一、顯微鏡下觀察薑黃素抑制抗原誘發RBL-2H3細胞脫顆粒反應……………………………………………………………………….76
圖十二、薑黃素對A23187誘發肥大細胞脫顆粒反應之影響 ……77
圖十三、顯微鏡下觀察薑黃素抑制A23187誘發RBL-2H3細胞脫顆粒反應 ………………………………………………………………78
圖十四、薑黃素抑制IgE媒介反應之組織胺釋放情形………………79
圖十五、薑黃素抑制A23187誘發RBL-2H3細胞組織胺釋放 ……80
圖十六、抗原誘發IgE媒介過敏反應中ROS之生成 ………………81
圖十七、薑黃素抑制IgE媒介過敏反應中ROS之生成 ……………82
圖十八、A23187誘發RBL-2H3胞內ROS生成 ……………………83
圖十九、薑黃素抑制A23187所誘發之ROS生成 …………………84
圖二十、薑黃素對RBL-2H3細胞受抗原刺激下catalase活性之影響………………………………………………………………………85
圖二十一、薑黃素對RBL-2H3細胞於IgE單獨致敏下catalase活性之影響…………………………………………………………………86
圖二十二、薑黃素對抗原誘發之PKC-�� 位移現象之影……………87
圖二十三、薑黃素抑制KU812之Fc�悐I 基因表現量 (12小時) ……88
圖二十四、薑黃素抑制KU812之Fc�悐I 基因表現量 (24小時) ……89
圖二十五、西方轉漬法分析薑黃素對Fc�悐I �� chain 蛋白表現量…90
圖二十六、薑黃素抑制KU812 之IL-4/IL-13基因表現量 …………91
表一、Fc�悐I 基因表現之PCR引子設計及序列產物長度…………92
表二、Th2細胞激素基因表現之PCR引子設計及序列產物長度…93














附錄

附圖一、薑黃素對抗各類疾病………………………………………94
附圖二、Th1及Th2細胞細胞激素之效用 …………………………..95
附圖三、細胞內過敏分子機制反應…………………………………..96
附圖四、肥大細胞被激發釋出介質示意圖……………………97
附圖五、鬱金類植物外型 ……………………………………………98
附圖六、薑黃素結構 …………………………………………………99
附圖七、肥大細胞中Fc�悐I媒介過敏反應之胞內訊息傳遞……100
附圖八、肥大細胞中Fc�悐I活化影響胞內鈣離子濃度變化……101
附錄九、過敏反應機制 ……………………………………………102
附錄十、 ROS產生與清除路徑 ……………………………………103
附表一、整理近年來薑黃素抗癌體外研究及其相關使用細胞株…104
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