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研究生:黃硯庭
研究生(外文):Yan-Ting Huang
論文名稱:以小孢子靈芝免疫調節蛋白治療誘導異位性皮膚炎大鼠的效用
論文名稱(外文):Effects of the Ganoderma microsporum Immunomodulatory Protein on Induced Atopic Dermatitis in Rats
指導教授:賴政宏賴政宏引用關係
指導教授(外文):Cheng-Hung Lai
口試委員:詹昆衛董光中
口試委員(外文):Kun-Wei ChanKwong-Chung Tung
口試日期:2019-05-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:87
中文關鍵詞:異位性皮膚炎小孢子靈芝免疫球蛋白E
外文關鍵詞:atopic dermatitisGanoderma microsporumIgE
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異位性皮膚炎(Atopic dermatitis; AD)是多重因素的疾病過程。它被定義為遺傳傾向的雙相炎症性和瘙癢性過敏皮膚疾病,通常會伴隨針對環境過敏原產生的免疫球蛋白E(IgE)。在異位性皮膚炎的致病機制中,主要被認為是全身系統性Th2細胞失衡,且伴隨著IgE的增加和嗜酸性球的增多。在疾病的急性期間可在皮膚中檢測到Th2細胞媒介的細胞激素,特別是IL-4、IL-5和IL-13。真菌免疫調節蛋白(Fungal immunomodulatory proteins; FIPs)具有誘導細胞激素表現的能力。小孢子靈芝免疫調節蛋白(G. microsporum immunomodulatory protein; GMI),是一種從小孢子靈芝中克隆(cloned)出來的真菌免疫調節蛋白,含有111個氨基酸,其氨基酸序列與其他FIP如LZ-8的氨基酸序列具高度同源性。證據顯示,純化和重組GMI具有顯著的抗發炎及抗腫瘤作用。在本次研究中,我們評估GMI對2,4-二硝基氯苯(2,4-dinitrochlorobenzene; DNCB)誘導大鼠異位性皮膚炎的抗發炎和抗過敏作用。將30隻雄性、7週齡Sprague-Dawley大鼠隨機分成6組,大鼠接受DNCB背部局部塗抹兩週,誘導異位性皮膚炎樣的皮膚病變,接著以GMI和prednisolone治療4週。結果顯示,在使用GMI治療後炎症細胞數量顯著減少(P <0.01),皮膚炎臨床症狀指數、表皮厚度、肥大細胞數量和免疫球蛋白E等數值,均呈現劑量依賴性的顯著降低(P <0.01)。此外,結果也顯示GMI具有與prednisolone類似的功效。基於以上這些證據,我們認為GMI確實可以改善DNCB所誘導的大鼠異位性皮膚炎,GMI是具有潛力作為過敏性疾病的輔助療法。然而,對於T細胞的調節機制以及GMI的安全性,仍需要進一步研究以制定理想的治療策略。
Atopic dermatitis(AD) is a multifactorial disease process. It is defined as a genetically predisposed biphasic inflammatory and pruritic allergic skin disease often associated with a production of immunoglobulin E (IgE) against environmental allergens. A predominant systemic Th2 disbalance with increased IgE levels and eosinophilia is widely accepted in the pathogenesis of atopic diseases. The production of Th2 mediated cytokines, notably IL-4, IL-5, and IL-13, can be detected in skin during the acute phase of disease. FIPs (Fungal immunomodulatory proteins) had the ability to induct cytokine expression. GMI (G. microsporum immunomodulatory protein), a FIP cloned from G. microsporum, contains 111 amino acids, with the amino acid sequence highly homologous with those of the other FIPs, including LZ-8. Evidence indicates that purified and recombinant GMI displays remarkable anti-inflammatory and anti-tumor effects. In this study, we assessed the anti-inflammatory and anti-allergic effects of GMI on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis in rats. Thirty male 7-weeks-old Sprague-Dawley rats were randomly divided into 6 groups. The rats received DNCB topically application on the back for two weeks to induce AD-like lesions, and then treated with GMI and prednisolone for four weeks. Our results demonstrated that the number of inflammatory cells were decreased after exposure of GMI (P <0.01), and the expression level of immunoglobulin E, dermatitis scores, epidermal thickness and mast cells were all reduced in a dose dependent manner (P <0.01). Additionally, our results showed that the GMI had similar efficacy to prednisolone. Based on this evidence, we believe that GMI could ameliorate the DNCB-induced atopic dermatitis in rats. It also possesses the potential to be used as an adjuvant therapy for allergic diseases. However, the mechanisms of the modulation on T cells and the safety of GMI need further studies for use as an ideal treatment strategy.
摘要……………………………………………………………………………… i
Abstract………………………………………………………………………… ii
目次…………………………………………………………………………… iii
表目次………………………………………………………………………… vi
圖目次………………………………………………………………………… vii
第一章 緒言………………………………………………………………… 1
第二章 文獻探討…………………………………………………………… 2
第一節 異位性皮膚炎………………………………………………… 2
一、 異位性皮膚炎簡介………………………………………… 2
二、 異位性皮膚炎致病機制………………………………… 2
三、 臨床上犬貓異位性皮膚炎之診斷……………………… 5
四、 臨床上犬貓異位性皮膚炎之治療(Olivry et al., 2010, 2015) ……………………………………………………… 8
五、 異位性皮膚炎誘導模組………………………………… 11
第二節 小孢子靈芝………………………………………………… 14
一、 靈芝…………………………………………………………… 14
二、 靈芝藥性成分與生理活性………………………………… 14
三、 小孢子靈芝免疫調節蛋白………………………………… 17
四、 小孢子靈芝藥性成分與生理活性……………………… 17
五、 真菌免疫調節蛋白(FIP)之萃取方式………………… 19
第三章 材料與方法……………………………………………………… 22
第一節 實驗動物…………………………………………………… 22
第二節 實驗材料…………………………………………………… 22
一、 藥物與試劑……………………………………………… 22
二、 儀器與設備……………………………………………… 23
三、 其他……………………………………………………… 23
第三節 實驗設計…………………………………………………… 24
一、 實驗分組………………………………………………… 24
二、 實驗設計流程…………………………………………… 24
三、 異位性皮膚炎實驗動物模型…………………………… 25
四、 萃取物之製備…………………………………………… 25
五、 評估異位性皮膚炎改善效用…………………………… 26
第四節 數據分析…………………………………………………… 29
第四章 結果………………………………………………………………… 30
第一節 體重變化………………………………………………………… 30
第二節 臨床症狀觀察………………………………………………… 35
第三節 皮膚炎臨床症狀指數評分…………………………………… 36
第四節 皮膚組織切片H&E染色之皮膚表皮層厚度…………… 46
第五節 皮膚組織切片H&E染色之炎症細胞浸潤程度………….. 49
第六節 皮膚組織切片TB染色之肥大細胞浸潤程度……………. 53
第七節 血清總IgE抗體檢測…………………………………………… 57
第八節 血清總IL-4檢測……………………………………………… 61
第五章 討論…………………………………………………………………… 65
第一節 異位性皮膚炎誘導效果評估……………………………… 65
第二節 大鼠實驗過程中的體重變化…………………………………… 65
一、 環境適應期………………………………………………… 65
二、 異位性皮膚炎誘導期………………………………………… 65
三、 異位性皮膚炎治療期………………………………………… 66
第三節 治療效果評估-皮膚炎臨床症狀指數評分……………………… 68
一、 環境適應……………………………………………………… 68
二、 異位性皮膚炎誘導期………………………………………… 68
三、 異位性皮膚炎治療期………………………………………… 68
第四節 治療效果評估-皮膚組織切片H&E染色及TB染色…… 69
一、 皮膚表皮層厚度…………………………………………… 69
二、 炎症細胞浸潤數量…………………………………………… 70
三、 肥大細胞浸潤數量…………………………………………… 71
第五節 治療效果評估-血清總IgE抗體及IL-4檢測………… 71
一、 血清總IgE抗體……………………………………………… 72
二、 血清總IL-4…………………………………………………… 73
第六節 比較類固醇與三種劑量小孢子靈芝在治療上的差異… 74
一、 類固醇……………………………………………………… 74
二、 小孢子靈芝免疫調節蛋白…………………………………… 75
第七節 影響本實驗治療的可能因素…………………………………… 76
一、 實驗動物品系……………………………………………… 76
二、 治療的頻率與療程………………………………………… 77
第六章 結論……………………………………………………………………… 78
參考文獻………………………………………………………………………… 79
一、 中文部分……………………………………………………… 79
二、 英文部分……………………………………………………… 79
附錄………………………………………………………………………………… 87
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