請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64424
標題: | 探討角質細胞spleen tyrosine kinase在IL-17引發CCL20表現所扮演的角色 Roles of spleen tyrosine kinase in IL-17-induced CCL20 chemokine expression in keratinocytes |
作者: | Hsin-Ni Tsou 鄒欣霓 |
指導教授: | 林琬琬 |
關鍵字: | 介白素第17A因子,脾酪氨酸蛋白激酶,角質細胞,CCL20趨化素, IL-17,Syk,keratinocytes,CCL20, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 介白素第17A因子(IL-17A)為第十七型輔助T細胞激素的其中一種,在自體免疫疾病的形成過程中扮演著重要的角色,諸如乾癬。根據現今越來越多證據指出抑制IL-17A對於治療乾癬具有顯著的臨床效果。脾酪氨酸蛋白激酶(Syk)為一種非受體性酪胺酸激脢,已知是多種免疫活化受體的重要訊息傳遞分子,因此現今被視為是多種自體免疫疾病的治療標靶。由於目前鮮少議題探討Syk在皮膚疾病及IL-17A訊息傳遞之間的關係。因此,本篇研究我們選用人類表皮角質細胞,以IL-17A刺激而誘發表現的趨化素CCL20作為研究模型,來探討Syk在IL-17受體之訊息傳遞的重要性,及參與表皮角質細胞的功能。我們發現IL-17A可依照時間及濃度的關係引發趨化素CCL20之基因的表現及蛋白質的釋出。並且在IL-17A刺激下,可以觀察到IKK,NF-kB,JNK,Syk之活化現象。藉由TAK抑制劑和Syk siRNA的處理,我們發現Syk為TAK訊息傳導之上游分子。Syk之抑制可顯著減少訊息激酶之活化及IL-17A誘導之趨化素CCL20表現。藉由結合啟動子活性檢測(promoter activity assay)及點突變之趨化素CCL20 reporter constructs的結果,我們更進一步證實此作用是源自於Syk所調控的NF-kB路徑。免疫沉澱分析的結果顯示在IL-17A之作用下,Syk會與IL-17受體的下游訊息傳遞成員,如TRAF6,Act1有交互作用之發生。但當Syk表現受到siRNA抑制時,TRAF6及Act1之交互作用將會減少。最後,我們利用免疫複合體做蛋白質激酶檢測(kinase assay),說明Syk能調控TRAF6之磷酸化。總體而言,我們首度發現Syk可作為IL-17A所導致Act1及TRAF6交互作用之上游訊息調控者,並且證實Syk在人類表皮皮質細胞中扮演著IL-17受體誘導NF-kB活化及趨化素CCL20基因轉錄等重要角色。這些發現不僅揭露Syk在IL-17A誘導發炎反應所扮演之嶄新角色,同時顯示出Syk足以成為治療乾癬之潛力標靶。 Interleukin-17A (IL-17A) is one of the Th17 cytokines and plays an important role in the immunopathogenesis of autoimmune diseases such as psoriasis. Up to date, accumulating evidence has strongly revealed the clinical benefits of inhibition of IL-17A for psoriasis treatment. Syk is a non- receptor tyrosine kinase and has been implicated as a critical mediator in various immune stimulation. Nevertheless, little is known about the relationship of Syk in skin disease and IL-17A signaling. Therefore in this study we used IL-17A-stimulated expression of CC chemokine ligand 20 (CCL20) in normal human epidermal keratinocytes as a cell model to investigate the role of Syk in this aspect. We found that IL-17A stimulation can induce CCL20 gene and protein expression in time- and concentration-dependent manners. Moreover, the activation of IKK,NF-kB, JNK and Syk were observed during IL-17A stimulation. By treating cells with TAK inhibitor and Syk siRNA, we found Syk is an upstream signal molecule of TAK. Inhibition of Syk strikingly attenuated all signal kinases activation and CCL20 secretion induced by IL-17A. Data of promoter activity assay combined with site-directed mutagenesis of CCL20 reporter construct further showed that IL-17A-elicited CCL20 upregulation is depending on Syk-mediated NF-kB pathway. Data using immunoprecipitation also indicated the interaction of Syk with IL-17R downstream signal components such as TRAF6 and Act1 under IL-17A stimulation. However, the essential signaling interaction of TRAF6 and Act1 under IL-17A stimulation was diminished when Syk expression was repressed by siRNA approach. Lastly, using immunocomplex kinase assay we demonstrated that Syk can mediate TRAF6 phosphorylation. Taken together, we for the first time identify Syk as an upstream signaling regulator in IL-17R-mediated Act1-TRAF6 interaction, and demonstrate that Syk plays an essential role for IL-17R-stimulated NF-kB activation and CCL20 gene transcription in primary human keratinocytes. All these findings not only unmask a new role of Syk in IL-17A-mediated inflammatory response, but also shed a new light into the potential therapeutic target of Syk in psoriasis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64424 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-101-1.pdf 目前未授權公開取用 | 1.41 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。