- 學位論文
青黴菌屬過敏原Pen c13誘導人類肺上表皮細胞其發炎性細胞激素與膜受器PAR-2相關性之探討
Penicillium sp. allergen induces inflammatory cytokines from human pulmonary epithelial cells: The serine protease allergen, Pen c13, activates protease-activated receptor (PAR-2)
摘要
黴菌為引起異位性疾病與IgE媒介之過敏疾病的重要致敏物種之一，研究顯示青黴菌 (Penicillium sp.) 和黃麴菌 (Aspergillus sp.) 為台灣地區的室內重要過敏原。當過敏病人吸入這些過敏原時，會產生過敏性鼻炎與氣喘等呼吸道疾病，但黴菌過敏原如何引發過敏反應，其詳細機制仍不清楚。先前的文獻報導發現，具有絲胺酸蛋白質水解酶 (serine protease) 活性的過敏原，可能透過肺部或支氣管上表皮細胞的膜受器PAR-2，引發一系列過敏相關之訊息傳導反應。因此，利用本實驗室先前所發現具有絲胺酸蛋白質水解酶活性的橘青黴 (P. citrinum) 過敏原Pen c 13，作為進一步探討相關致敏機制的研究材料。 以pQE30載體系統轉殖到大腸桿菌M15中，大量表現重組蛋白質rPen c 13，再經由親和性管柱進行純化，以此進行免疫轉漬實驗，發現25% 的病人血清對rPen c 13具有IgE結合能力。對這些篩選出的病人予以抽血並以天然型Pen c 13 (nPen c13)進行組織胺釋放實驗，結果顯示nPen c 13確實具有刺激過敏病人血球細胞釋放出組織胺的能力。 以nPen c 13刺激人類肺表皮細胞株A549與透過RT-PCR的偵測，發現許多前發炎反應細胞激素、化學激素 (GM-CSF、IL1β、IL-6、IL-8) 的mRNA表現量都有增加。除基因表現層次之外，也利用ELISA實驗進行相關細胞激素在蛋白質層次上的檢測，發現與發炎反應有關的IL-8與IL-6有顯著的上升，而且其上升幅度和nPen c 13呈現劑量相關性 (dose-dependent)，當nPen c 13濃度分別為0.1、0.05 μg/mL時，IL-8與IL-6達到最大表現量。由上述實驗證實，nPen c 13可以引發人類肺表皮細胞株A549產生發炎相關反應。為進一步探討Pen c13對A549造成的發炎反應，是否與其絲胺酸蛋白質水解酶的活性有關，我們選用絲胺酸蛋白質水解酶抑制劑：PMSF、AEBSF與nPen c 13進行反應，發現反應後的nPen c13無法刺激A549細胞株產生IL-8表現量的上升。 另外，我們也試著探討nPen c13對A549造成的發炎反應，是否與膜受器PAR-2有關。因此在進行nPen c13對A549的刺激反應前，先以PAR-2單株抗體阻絕A549膜上受器PAR-2；結果顯示A549中IL-8的表現量有下降的情形。接著我們以無鈣離子緩衝液處理細胞，發現IL-8的量也大幅下降，發現鈣離子的有無對PAR-2所主導的訊息傳遞途徑很重要。 本研究證實nPen c13的確能刺激組織胺的釋放，與誘發肺部上表皮細胞的發炎反應。nPen c 13誘發發炎反應的能力與其絲胺酸蛋白質水解酶活性有關，而且可能是透過膜受器PAR-2產生的結果。以上與前人對塵蟎敏原與PAR-2相關性的研究結果相似。透過對過敏分子機制的了解，未來在呼吸相關過敏疾病的治療與藥物的開發上，可提供許多有用的資訊。
關鍵字
絲胺酸蛋白質水解酶並列摘要
The fungi are regarded as the main source of allergens that can cause IgE-mediated allergic and atopic disease. Penicillium and Aspergillus are the most common indoor fungal species in Taiwan. It has been recognized that inhalation of fungal allergens can produce allergic rhinitis and asthma in susceptible individuals. But the mechanism about how these allergens induce allergic reaction is still not clear. Recent studies have shown the existence of PAR-2 (proteinase-activated receptor 2) which is expressed on the bronchial and respiratory epithelium cells may be activated by proteolytic allergens. Thus we used the allergen Pen c13, the serine protease we had purified from P. citrinum, to investigate the mechanism of allergy. The coding cDNA of Pen c13 was cloned into vector pQE30 and expressed in E. coli M15 as a His-tag fusion protein and purified by affinity column. The rPen c13 reacted with serum IgE from 25% of allergic patients. Purified nPen c13 elicited dose-dependent basophil and mast cell histamine release in patients allergic to Penicillium. In the pulmonary epithelial cell line A549, GM-CSF, IL1β, IL-6 and IL-8 mRNA levels were enhanced by nPen c13. Furthermore, ELISA was also used to detect IL-8 and IL-6 in protein level. Both of them showed significant release after the stimulation by nPen c13 in a dose-dependent manner. The maximum concentrations which IL-8 and IL-6 released were observed at 0.1ug/ml and 0.05 ug/ml respectively. From these experiments we conclude that nPen- c13 can induce inflammatory reaction in A549. Serine protease inhibitors, PMSF and AEBSF, were then chosen to inactivate nPen c13 in order to check whether the inflammatory reaction was caused by nPen c13. The result obtained indicated that IL-8 stimulated by nPen c13 was abolished when protease activity was inhibited by PMSF and AEBSF. Furthermore, Western blot analysis showed that COX-2 expression was also enhanced by nPen c13. Additionally, we tried to understand whether PAR-2 involved in this inflammatory reaction of A549 caused by nPen c13, antibody against the cleavage region of PAR-2 was used in order to block PAR-2 cleavage by nPen c13. The result indicated the expression of IL-8 was reduced in A549. Then cells were treated with Ca2+ free buffer and the production of IL-8 by nPen c13 significantly decreased, so Ca2+ may play an important role in the inflammatory reaction induced by PAR-2. The present study indicated that nPen c13 can indeed stimulate histamine release and induce inflammatory reaction in respiratory epithelium cells. The latter reaction of nPen c13 involves its own serine protease activity and PAR-2 may play a role in this result. Our findings are similar to previous studies of mite allergen with PAR-2. Advances in understanding the molecular mechanism of atopy may lead to new therapies for allergic diseases in the future.