江伯倫臺灣大學:口腔生物科學研究所簡芊卉Jian, Chien-HueiChien-HueiJian2010-05-042018-07-092010-05-042018-07-092009U0001-1708200914431100http://ntur.lib.ntu.edu.tw//handle/246246/178618氣喘是兒童常見的過敏性疾病之一，根據統計全球約一成的人口有氣喘的困擾，歐洲塵蟎 (Dermatophagoides pteronyssinus) 是八成台灣過敏病患的過敏原之一。氣喘的臨床症狀包含：呼吸道過度反應、嗜酸性白血球浸潤於呼吸道、活化的過敏原特異性第二型輔助型T細胞增加、呼吸道黏液增加、甚至是呼吸道重塑等等。口服耐受性，是指透過口服抗原而引發抗原特異性的免疫反應下降的現象，目前被認為具潛力成為過敏性氣喘的治療方式。本篇研究希望透過口服餵食基因重組第二類與第一類歐洲塵蟎過敏原 (recombinant D. pteronyssinus group 2 allergen, rDp2; recombinant D. pteronyssinus group 1 allergen, rDp1)，藉此來降低由歐洲塵蟎粗萃蛋白所引發的呼吸道發炎現象。我們利用腹腔注射粗萃蛋白致敏BALB/c雌性小鼠，並給予氣管粗萃蛋白引發呼吸道發炎。結果顯示，腹腔注射高劑量 (50 μg) 的粗萃蛋白可有效誘發過敏性氣喘的臨床症狀，包含血清中塵蟎蛋白特異性的IgE含量增加、促進嗜酸性白血球浸潤於肺部、肺部沖洗液中介白素-5 (Interleukin-5, IL-5) 含量增加、小鼠脾臟細胞經粗萃蛋白刺激可產生大量Th2的細胞激素 (IL-5與IL-13) 並且大量增生。更進一步，我們利用口服餵食的方式，連續七天給予致敏小鼠0.2或1.0 mg劑量的rDp2或粗萃蛋白，探討是否能有效降低粗萃蛋白引發的氣喘症狀。結果發現，給予1.0 mg的rDp2可有效降低呼吸道過度反應，並減緩呼吸道發炎現象。因此，我們認為餵食較高劑量的基因重組蛋白或粗萃蛋白，有減緩過敏性氣喘症狀的傾向；未來將需要更多的實驗，以尋找合適的餵食計量與時間。Asthma is one of the most common allergic diseases in children; in addition, about 80% of asthmatic patients in Taiwan are sensitized by house dust mite -- D. pteronyssinus. The characteristics of asthma such as AHR, eosinophils infiltration, antigen-specific T helper 2 cells activation, increased mucus secretion and even airway remodeling. Antigens-specific immune tolerance by prior oral administration of antigens might be a therapeutic strategy for allergic asthma. Therefore, we aimed to apply oral administration of rDp2 to decrease the airway inflammation induced by D. pteronyssinus. The female BALB/c mice were used and given with crude mite extract of D. pteronyssinus as the allergic asthma. In present study, we sensitized mice with peritoneal injection, and then challenged with intratracheal injection of crude mite extract. The results showed that peritoneal injection with high-dose could induce the clinical features of asthma significantly, including elevated mite-specific IgE in serum, production of Th2 cytokines (IL-5, IL-13) of splenocytes, and crude mite-specific lymphoproliferation. Furthermore, oral delivery of rDp2 or crude mite extract 0.2 or 1.0 mg/day for consecutive 7 days at the beginning of sensitization showed some beneficial effects on airway inflammation. Oral feeding 1.0 mg/day of rDp2 reduced AHR and slightly decreased the airway inflammation induced by crude mite extract. In conclusion, we suggest that oral delivery of high dose of single recombinant allergen seems to be more benefit on airway inflammation induced by the complex crude mite extract, and the feeding dose and feeding period need further investigation.Brief Contents文摘要 …………………………………………………………………… Ibstract …………………………………………………………………… IIrief Contents …………………………………………………………… IVull Contents ………………………………………………………… Vontents of Figures ………………………………………………………VIIIbbreviation ……………………………………………………………… IXntroduction ………………………………………………………… 1ypothesis & Specific Aims ………………………………………… 11aterials and Methods ……………………………………………… 13esults ………………………………………………………………… 28iscussion & Conclusion …………………………………………… 40igures ……………………………………………………………… 49eferences ……………………………………………………………70ppendix …………………………………………………………… 80ull Contents ntroduction ………………………………………………………………………… 1pidemiology of allergic asthma ……………………………………………… 2isk factors for asthma ………………………………………………………… 2efinition of asthma …………………………………………………………… 4echanism of asthma …………………………………………………………… 4nimal model for allergic asthma ……………………………………………… 6rugs for asthma treatment ……………………………………………………… 6ral tolerance …………………………………………………………………… 7ouse dust mite ………………………………………………………………… 9ecombinant allergens ………………………………………………………… 11ypothesis & Specific Aims ………………………………………………………… 11aterials and Methods ……………………………………………………………… 13reparation of crude mite extract ……………………………………………… 14reparation of rDp2 …………………………………………………………… 14xpression of rDp1 …………………………………………………………… 15estern blotting of rDp2 and rDp1 …………………………………………… 18nimals ………………………………………………………………………… 19ensitization and challenge of crude mite extract to establish allergic airway inflammation …………………………………………………………………… 19etermination of levels of crude mite specific antibodies in serum …………… 20etermination of levels of total IgE in serum ………………………………… 21easurement of AHR ………………………………………………………… 23nalysis of cellular composition of BALF …………………………………… 23etermination of cytokines secretion of splenocytes by ELISA ……………… 24etermination the lymphoproliferation of splenocytes ………………………… 26ral feeding protocol ………………………………………………………… 27tatistical analysis ……………………………………………………………… 27esults ……………………………………………………………………………… 28uccessful expression of rDp2 from a fermentor produced by Pichia pastoris .. 29xpression of rDp1 protein produced by Pichia pastoris ………………… 29ensitization with crude mite extract elevated specific immunoglobulin ……… 31ensitization and challenge with crude mite extract increased lymphoproliferation of splenocytes ………………………………………………………………… 32ensitization and challenge with crude mite extract increased inflammatory cytokine secretions of splenocytes …………………………………………… 33ensitization and challenge with crude mite extract induced AHR …………… 33ensitization and challenge with crude mite extract facilitated eosinophils infiltration in BALF …………………………………………………………… 34ral feeding showed no effects on the levels of immunoglobulin in serum …… 35he lymphoproliferation were no affected by oral feeding with mite allergens .. 36L-4 productions of splenocytes decreased after oral feeding ………………… 36ral feeding with mite allergens slightly elevated IL-10 levels of splenocytes .. 37ral feeding with higher dose of mite allergens reduced the AHR …………… 38he airway inflammation was slightly decreased after oral feeding …………… 38iscussion & Conclusion ………………………………………………………… 40llergic asthma model induction ……………………………………………… 41he effects of oral delivery with rDp2 on airway inflammation ……………… 43he effects of oral delivery with crude mite extract …………………………… 46onclusion ……………………………………………………………………… 47igures ……………………………………………………………………………… 49eferences ………………………………………………………………………… 60ppendix ………………………………………………………………………… 80ppendix 1. Schematic representation of vector maps of rDp1 or rDp2 ……… 81ppendix 2. Sequence of pPICZα-A-der p 1 ………………………………… 82ppendix 3. Reagents ………………………………………………………… 83application/pdf7102910 bytesapplication/pdfen-US過敏性氣喘歐洲塵蟎第二類歐洲塵蟎過敏原口服耐受性allergic asthmaDermatophagoides pteronyssinusDp2oral tolerancehttp://ntur.lib.ntu.edu.tw/bitstream/246246/178618/1/ntu-98-R96450005-1.pdf