在小鼠氣喘模式中以口服給予Der p 2過敏原誘導抗原特異之耐受性

Abstract

中文摘要 氣喘是目前在兒童最普遍發生的過敏疾病，在開發中及已開發國家中約有百分之十的兒童面臨此種疾病。過敏性氣喘往往伴隨著呼吸道過度反應、嗜酸性白血球在肺部的浸潤、呼吸道分泌過多黏液等症狀。口服給予某種特定抗原可以導致免疫系統對於此種抗原的不反應現象，稱之為「口服耐受性」。口服耐受性目前已知的機制是肇因於誘導T淋巴球對於特定抗原的不反應、或排除對特定抗原有免疫反應的T淋巴球、或是免疫系統活化一群調節性T淋巴球。在對於過敏性疾病的治療方面，口服耐受性的應用似乎是一種可行的方法。誘發口服耐受性需要大量口服給予蛋白質，若是用以往獲得過敏原蛋白的傳統方法，如直接從塵、植物、動物萃取過敏原蛋白而來，不但會有其他雜蛋白污染的問題，成本較為昂貴且產量低。蛋白質重組技術可以解決以上的問題。我們使用了酵母菌Pichia pastoris蛋白質表現系統，來表現主要過敏原「家塵Dermatophagoides pteronyssinus第二群過敏原」(Der p 2)。然後，以酵母菌生產的Der p 2 餵食氣喘模式的小鼠，以評估誘發口服耐受性對於治療過敏性氣喘疾病的實用性。此外， 我們也建立另一種重組蛋白系統Der p 2基因轉殖番茄植株來大量表現Der p 2 重組蛋白質。 在餵食低劑量由酵母菌生產而來的Der p 2過敏原於此過敏原致敏的小鼠動物模式中，研究發現可以降低對於Der p 2過敏原有專一性的免疫球蛋白IgE與IgG1的產生，其呼吸道過度反應的現象相對於餵食緩衝液的正對照組也較緩和。 而分析肺沖洗液的實驗中發現小鼠餵食重組Der p 2過敏原後，其呼吸道的嗜酸性白血球浸潤現象程度降低。但在全身性耐受指標方面，如脾臟細胞對於過敏原的刺激反應或是其細胞激素的分泌，並沒有明顯的差異。 總論而言，此研究顯示口服低劑量酵母菌生成的Der p 2 過敏原在小鼠氣喘模式中可誘發口服耐受性。未來，應用口服Der p 2基因轉殖番茄誘發口服耐受性以治療氣喘疾病是可以期望的。

Abstract Asthma is one of the most common allergic diseases in children, and affects more than 10% children population in both developing and developed countries. Allergic asthma is associated with airway hyperresponsiveness (AHR), eosinophils infiltration and increased mucus secretion in the airway. Oral administration of antigen is a method of inducing antigen-specific unresponsiveness that is called oral tolerance. That is result of inducing antigen-specific T cells anergy or deletion or a group of antigen-specific regulatory T cells to suppress antigen-specific immune response. The induction of oral tolerance is suggested to be a possible immunotherapy for allergic diseases. A large amount of allergen is needed for oral administration to induce oral tolerance. However, the traditional method to generate a specific allergen from natural mites, plant or animal extracts is limited due to the problem of other unnecessary molecules contamination and expensive but low-yield production. Recombinant technologies can be used to prevent these problems. We hope to develop a large scale protein expression system for allergen specific immunotherapy. In the present study, we used the yeast Pichia pastoris protein expression systems for producing the major allergen, house dust mite Dermatophagoides pteronyssinus group 2 allergen (Der p 2). Then, the recombinant Der p 2 proteins derived from yeast was fed to a Der p 2-sensitized asthma murine model to investigate the application of oral tolerance induction as therapy for allergic asthma. Furthermore, we also generated a protein expression system of Der p 2-transgenic tomato plant to produce large amount of rDer p 2. In this study, oral feeding with low dose of rDer p 2 derived from yeast would decrease Der p 2-specific IgE and IgG1 titers in serum after immunization. The airway hyperresponsiveness was significantly reduced in mice fed with rDer p 2 compared to the positive control. The infiltration of eosinophils in BALF was also decreased in mice fed with rDer p 2. Those results indicated that mice fed with rDer p 2 could inhibit the local airway inflammation. However, the systemic immunological tolerance indexes, such as the proliferation and cytokine secretion upon allergen stimulation of splenocytes, did not show significant difference. Conclusively, our data suggested that oral delivery of low dose yeast-derived rDer p 2 could induce oral tolerance in a local allergic airway inflammation murine model. The application of oral delivery of Der p 2-transgenic tomato to induce oral tolerance as a new therapy for asthma diseases is prospective.