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研究生:李志鴻
研究生(外文):Jyh-Hong Lee
論文名稱:蘭格素Langerin (CD207) 所媒介的過敏性發炎反應在異位性皮膚炎之研究
論文名稱(外文):In Vivo Imaging Reveals that Langerin Mediating Uptake of Ovalbumin is Associated with the Allergic Inflammation in Atopic Dermatitis
指導教授:江伯倫江伯倫引用關係
口試委員:王莉芳董成淵李婉若高甫仁
口試日期:2012-12-27
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
語文別:中文
論文頁數:164
中文關鍵詞:異位性皮膚炎蘭格素蘭格罕細胞經皮致敏鉻貴橄欖石飛秒脈衝雷射-多模式非線性光學倍頻顯微術
外文關鍵詞:atopic dermatitisLangerhans cellsLangerin (CD207)epicutaneous sensitizationCr:forsterite laser-based multi-modality nonlinear microscopy
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異位性皮膚炎 (Atopic dermatitis, AD) 屬於慢性的發炎性疾病,它的主要特色之一就是反覆的發作,在異位性皮膚炎的急性變化裡,表皮層的主要特徵是角質層的過度角質化與表皮細胞的增生,在異位性皮膚炎的慢性變化裡,真皮層的主要特徵是苔蘚化與纖維化。我們首先建立異位性皮膚炎小鼠動物模式,然後利用鉻貴橄欖石飛秒脈衝雷射-多模式非線性光學倍頻顯微術 (Cr:forsterite fs laser-based multi-modality nonlinear microscopy) 來評估是否可以不用皮膚切片而可以進行異位性皮膚炎的活體評估。我們發現三倍頻訊號 (third-harmonic generation signal, THG signals) 所呈現的皮膚內表皮層的表皮細胞與角質層的增厚,我們也可以觀察到二倍頻訊號 (second-harmonic generation signal, SHG signals) 所呈現的真皮層中的膠原蛋白纖維的纖維化,我們進一步發現,角質層中雙光子螢光訊號 (two-photon fluorescence signals, TPF) 的強度與異位性皮膚炎的嚴重度有關。
已知異位性皮膚炎和皮膚對於環境中的抗原引發之過敏反應有關,但是它的詳細致病機轉,特別是過敏原如何被呈現,都是重要卻懸而未決的問題。已知過敏原的呈現與C-type lectins receptors (CLRs) 有關,所以我們希望以此方法去瞭解過敏原被呈現而致敏的過程。我們主要採用鉻貴橄欖石飛秒脈衝雷射-多模式非線性光學倍頻顯微術來從事相關的研究,並且利用適合的單株抗體來標定、追蹤以及阻斷或修飾相關CLRs,特別是蘭格罕細胞其細胞表面的蘭格素 [Langerin (CD207)] 分子的作用。我們的結果指出經皮致敏的過程中,表現蘭格素的蘭格罕細胞發生從皮膚換位移動到淋巴結。我們的結果也指出經皮致敏的過程中,蘭格素的表現量的變化反映出蘭格罕細胞在經皮致敏的狀態下接受OVA抗原的刺激而產生功能成熟的表現型。我們的結果也顯示結合Alexa Fluor 647與鉻貴橄欖石飛秒脈衝雷射-多模式非線性光學倍頻顯微鏡兩種工具,我們可以偵測到皮膚與淋巴結內的抗原或過敏原,或是攜帶抗原或過敏原的細胞。
我們假設OVA抗原經由蘭格素進行吸收終究導致Th2 T淋巴球的活化,進而引起異位性皮膚炎的慢性發炎反應。因此,我們採用單株抗體去阻斷蘭格素相關的抗原呈現以及免疫調控的作用,我們將比較異位性皮膚炎的皮膚組織學以及免疫血清學的變化。我們的數據顯示在第三天至第五天,接受阻斷性抗體的老鼠血清中total IgE的濃度降低與經腹注射針對蘭格素的阻斷性抗體有關。我們的結果顯示經腹注射針對蘭格素的阻斷性抗體也會減輕異位性皮膚炎的小鼠動物模式中,皮膚組織學上明顯的角質層與表皮層增厚與真皮層纖微化,暗示著蘭格素在異位性皮膚炎的皮膚局部發炎反應是必須的。我們的體外實驗結果顯示類蘭格罕細胞的吸收白蛋白 (OVA, ovalbumin) 必須依賴包括蘭格素在內的CLRs。
我們認為鉻貴橄欖石飛秒脈衝雷射-多模式非線性光學倍頻顯微術是一種安全的活體成像技術,可以應用於臨床追蹤及基礎研究,也可以促進我們去實際瞭解活體中異位性皮膚炎的詳細致病機轉,研發真正針對特定分子細胞的疾病治療策略,有助於找出以免疫調控為理論基礎的異位性皮膚炎長期控制之道。

Atopic dermatitis (AD) is characterized by hyperkeratosis of epidermis and fibrosis within dermis in chronic skin lesions. Thus far, the histology of skin lesions has been evaluated only by examination of excised specimens. We used Cr:forsterite laser-based multi-modality nonlinear microscopy to analyze the endogenous molecular signals, including third-harmonic generation (THG), second-harmonic generation (SHG), and two-photon fluorescence (TPF) from skin lesions in AD. Significant differences in thickness of epidermis and stratum corneum (SC) and modified degrees of fibrosis in dermis (measured by THG signals and SHG signals, respectively) were clearly demonstrated in in vitro studies. Increased TPF levels were positively associated with the levels of the THG signals from the SC. These findings were reproducible in skin lesions from human AD. Our findings suggest that the optical signatures of THG, TPF, & SHG can be used as molecular markers to assess the pathophysiological process of AD and the effects of local treatment.
It remains unclear that CD207 of Langerhans cells (LCs) play a central role in the development of allergic sensitization. We used a murine model of epicutaneous (EC) ovalbumin (OVA) sensitization inducing an inflammatory skin resembling AD to explore the role of CD207 in the pathogenesis of AD. Cr:forsterite laser-based multi-modality nonlinear microscopy was applied for in situ imaging. Peritoneal injections of Alexa Fluor 647-rat anti-mouse CD207 into mice were performed to specifically trace the LCs. Peritoneal injections of OVA-Alexa Fluor 647 conjugate into mice were performed to specifically trace the OVA. We found that combining Alexa Fluor fluorescent probes with multi-modality nonlinear microscopy permitted the unequivocal in situ imaging of CD207-expressing LCs. The relevant time-course, expressional and functional studies revealed that CD207 of LCs play an essential role during the induction of EC sensitization. It’s suggested that CD207-expressing LCs initiate the allergic response through the CD207 mediated EC sensitization associated with the development of AD.
AD patients show increased total serum IgE levels. The anti-IgE therapy has only limited efficacy in treating AD so that a prerequisite role of Langerhans cell (LC) in the development of AD were explored. We induced an animal model of AD by epicutaneous sensitization with ovalbumin (OVA). Peritoneal injection of blocking mAb against CD207 was given before epicutaneous sensitization and serum level of total IgE and skin histology were analyzed. The levels of total IgE and the epidermal thickness from mice group received blocking mAb were significantly reduced than those did not. The levels of plasma IL-4 and OVA-specific IgE in response to OVA sensitization in sensitized mice treated with blocking mAb anainst CD207 in vivo was lower than that from mice without blocking treatment. Our studies demonstrated that CD207 antagonists could attenuate allergic inflammation. We suggest that OVA uptake process associated with CD207 might contribute to the allergic response and play a role in the pathogenesis of AD.

誌謝……………………………………………………………………p. i

中文摘要……………………………………………………………p. ii

英文摘要……………………………………………………………p. iv

目錄…………………………………………………………………p. vi

圖說目錄………………………………………………………… p. vii

緒論 (INTRODUCTION) …………………………………………p. 1

研究材料與方法 (MATERIALS & METHODS) ……… …… p. 21

結果 (RESULTS) ………………… ………………………………p. 27

討論 (DISCUSSION) ………………………………… ………… p. 43

展望 (PROSPECTIVES) …………………………………………p. 61

論文英文簡述 (ENGLISH SUMMARY) ……………………… p. 71

參考文獻(REFERENCES)…………………………………… p. 89

圖說 (FIGURES & LEGENDS)…………………………………p. 109

主要論文………………………………………… ………………p. 163


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