主要過敏原Der f2刺激A549細胞與EBV轉型B淋巴球之蛋白體研究與免疫特性分析

第一部分 氣喘是嚴重的呼吸道疾病之ㄧ，但是至今其詳細的致病機制仍未清楚。引發氣喘的原因有很多種，目前認為可能的原因之一是經由呼吸道過敏所引發。空氣中的過敏原已被證實是引起氣喘的原因之ㄧ，其中塵螨為最重要的室內過敏源，是吸入性過敏原的主要來源之ㄧ。目前被鑑定出的塵螨過敏原可分為19群，多達十餘種以上。其中第一群與第二群塵螨過敏原於過敏血清的IgE反應性最高，皆為80-100%。 第一群塵螨過敏原被鑑定為半硫胺酸蛋白酶 (cysteine protease)，其蛋白酶活性被證實與其致敏機制有關。然而第二群塵螨過敏原，其蛋白質特性與致敏機制卻仍不清楚。因此，本研究利用純化之重組第二群塵螨過敏原Der f 2 (DF2) 刺激呼吸道上皮細胞A549以及分離自過敏病人之B淋巴球，進行蛋白體分析與免疫特性分析並企圖進一步發現第二群塵螨過敏原的分子致病機制。 在本實驗中，我們發現DF2會抑制A549細胞分泌IL-8，這與先前DF1的研究報導剛好相反。接著利用MTT的方式分析發現經由DF2刺激後會降低A549細胞的貼附性並促進其分離，但目前並未有文獻報導DF2 具有蛋白酶活性。此外，以不同的蛋白酶抑制劑不會影響DF2降低A549細胞貼附性的能力。因此，我們推測DF2降低A549細胞貼附性並不是透過蛋白酶的活性。最後，我們企圖透過二維電泳分析，進行DF2刺激對A549蛋白質表現的影響，並對具有顯著差異性的蛋白質進行質譜分析與蛋白質身份鑑定。 總而言之，本研究是首度以蛋白體研究法與免疫特性分析去探討具有高度致敏性的第二群塵螨過敏原之分子致病機制。透過對此一機制的研究，不僅可以幫助我們去瞭解DF2的蛋白質特性與可能的致敏機制，更可以作為發展新的減敏治療標的之重要的前驅性研究。第二部分 過敏和自體免疫皆為常見的免疫系統疾病，近年來越來越多的研究指向這兩種疾病之間有著某種關連性。除此之外，我們在臨床上發現有些病人同時罹患紅斑性狼瘡和過敏性氣喘這兩種疾病，因此讓我們更加確定這兩種疾病之間也許存在某些未知的關連性。 為了要探討這兩種疾病之間的關係，我們抽取這一類病人的血液之後，將B細胞分離出來，再利用Epstein-Barr virus來進行轉型作用，使得B細胞變成不朽的狀態，以利進行接下來的研究。 我們以塵螨過敏原DF2處理這些B細胞之後，再以蛋白質體的方式分析B細胞的蛋白質表現量。在處理之後，我們發現有5個蛋白質的表現量有顯著的下降，最後利用LC-MS/MS鑑定這五個蛋白質。 鑑定的結果發現這五個蛋白分別為Uncharacterized protein C17orf59、Myozenin-3、Profilin、Alpha-Enolase和Glutathione S-transferase。其中Alpha-Enolase 和 Glutathione S-transferase已被證實為自體抗原。本篇的實驗結果可做為日後研究過敏和自體免疫的根據。

Part 1 Asthma is one of the life-threatening allergic diseases around the world. However, the molecular pathophysiological mechanisms of asthma are not well-known. Airborne allergens have been demonstrated that may induce or trigger the development of asthma, but the molecular pathogenesis still remains unclear. House dust mite (HDM) is one of the most important sources of indoor aeroallergens; it contains 45 identified allergens which has be classified into 19 groups. Among the identified allergens of HDM, group I- and group II-allergen shows the highest frequency of IgE-reactivity. Group I-HDM allergen, belonging to cysteine protease, has been reported that protease activity significantly contributes to its allergenicity and pathogenesis. Interestingly, group II-HDM allergen, showing the high frequency of IgE-reactivity as group I-HDM allergen, has few studies for its protein characterization and pathogenic mechanisms. In the present study, we aimed to investigate the pathogenic mechanisms induced by group II-HDM allergen. With treatment of a group II-HDM allergen Der f 2, the culture medium and the treated epithelial cell A549 were collected individually for immunoassays and gel electrophoresis. By using MTT assay, two-dimensional gel electrophoresis, IL-8 production assay, dot blotting assay, ELISA and immunoblotting assay, the effects of Der f 2 on A549 has been investigated. According to the results, Der f 2 significantly decreased the adhesion and increased the dissociation of A549 cell. Der f 2 also induced a differential protein profile on 2-D gel. The proteins significantly changed on 2D gel by DF2-treated were further identified by peptide-mass fingerprinting (PMF). Our result show DF2-treatment may alleviate the cell adhesion and increase the cell dissociation of A549 cell. Note worthily, DF2-treatment may decrease the IL-8 production for A549 cell. On the contrary, group I-HDM allergen such as Der p 1 or Der f 1, has been found increased the IL-8 production for A549. In conclusion, this study first demonstrated that DF2 may reduce the cell attachment to basement, alter the protein expression and decrease the IL-8 production for A549 cell line. The findings may not only provide a possible pathway triggered by group II-HDM allergen, but also reveal a new strategy for therapeutic treatment in airway hypersensitivity.Part 2 Autoimmune and allergy are both common immune disease, but they are immunologically defined as contrary events resulting from TH1 and TH2 response. However, more and more evidences show that autoimmune and allergy have important correlations. In order to investigate the correlation between autoimmune and allergy, we obtained B lymphocytes from patient suffering SLE and allergic asthma and immortalized the B cells by Epstein-Barr virus transformation. After challenged with a major aeroallergen, Der f2, the immortalized B cells were lysed and followed two-dimensional gel electrophoresis (2-DE). Many regulated proteins were presented and several of them further identified by peptide sequencing using LC-MS/MS. In summary, we identified and characterized several important proteins induced by Der f2 allergen, and the down-regulated proteins including Uncharacterized protein C17orf59、Myozenin-3、Profilin、Alpha-Enolase and Glutathione S-transferase。Alpha-Enolase and Glutathione S-transferase were reported as autoantigens. Our findings should be useful in unveiling the known molecular mechanism between allergy and SLE development.