Objective:To optimize the challenge scheme for establishing a stable mouse model of Artemisia annua pollen-induced allergic rhinitis. Methods:BALB/c mice were subcutaneously injected with 0.1 ml allergen extract containing 20 μg/ml Art a1 from Artemisia pollen on 1 d, 4 d and 7 d. One week after the sensitization, these mice were divided into three groups and intranasally challenged with Artemisia annua pollen allergen extract containing 500 μg/ml Art a1 for 7 (7 d group), 10 (10 d group) and 14 (14 d group) consecutive days, respectively. The first challenge was followed by another 7 days of challenge every four weeks. Blank control group was set up through sensitizing and challenging BALB/c mice with normal saline. Behavioral changes and nasal pathological changes were observed. The changes in humoral and cellular responses were also detected. After the first challenge cycle was decided, the challenge frequency was further optimized. Results:After the first challenge, the allergic symptoms of mice in 10 d group were significantly severe than those in 7 d and 14 d groups, and the levels of serum specific IgE antibody in 10 d and 14 d groups were significantly higher than that in 7 d group. After the second challenge, the mice in the three model groups still had obvious allergic symptoms as compared with the blank control group. There were obvious pathological changes in the nose, including epithelial cell proliferation, turbinate enlargement and inflammatory cell increase. Moreover, the level of serum specific IgE antibody increased significantly and the proliferation of antigen-specific IL-4 and IL-6 lymphocytes was significantly up-regulated, especially in 10 d and 14 d groups. The frequency of challenge had a great impact on the stability of the allergic model. The allergic symptoms of sensitized mice challenged every two weeks were significantly severe than those of mice challenged every four weeks and the level of serum antigen-specific antibody was also higher.Conclusions:This study optimized the first challenge cycle and challenge frequency for establishing a mouse model of Artemisia annua pollen-induced allergic rhinitis, which provided reference for the establishment of drug efficacy evaluation system for desensitization therapy.

目的 基于屋尘螨变应原组主要变应原(Der.p1)的剂量建立稳定的屋尘螨过敏小鼠模型,并以此系统筛选过敏评价生物标识.方法 以屋尘螨变应原提取物为致敏蛋白,以氢氧化铝为佐剂,依据主要变应原(Der.p1)含量设置不同抗原浓度组和不同免疫针次,皮下注射免疫BALB/c小鼠,末次免疫一周后,采用Der.p1浓度为500μg/mL的屋尘螨变应原提取物滴鼻激发,每只小鼠20μL,每天1次,持续1周.通过观察小鼠的过敏反应症状、检测鼻部组织病理变化、测定各组小鼠体液免疫和细胞免疫指标的变化,筛选最佳的过敏模型建立程序.在此基础上更换不同来源屋尘螨抗原重新致敏BALB/c小鼠,验证过敏小鼠模型建立方法的可重复性.结果 致敏小鼠经抗原激发后均出现明显的过敏反应,其中最佳免疫程序为Der.p1浓度100μg/mL、皮下注射两次.该致敏组小鼠经抗原激发后体内IL?4特异性淋巴细胞数(174±23)明显增多,与阴性对照组(22±0.5)比较差异具有统计学意义(P=0.011);脾淋巴细胞Th2型极化显著(Th1/Th2比值为0.362±0.028),与阴性对照组(0.832±0.07)比较差异具有统计学意义(P=0.034);血清屋尘螨特异性IgE抗体水平与激发前比较明显增高(P=0.00017),总IgE抗体水平与阴性对照组比较明显增高(P=0.041).利用不同来源的屋尘螨抗原,选择优化的致敏程序建立过敏小鼠模型,表现出较好的可重现性.结论 成功构建屋尘螨变应原特异性致敏小鼠模型,并以此筛选到致敏相关的生物标识,证实Der.p1剂量与致敏效果间的量效关系,为后续屋尘螨过敏症机制研究及屋尘螨变应原脱敏制剂研制提供实验基础.

Objective:To optimize the BALB/c mouse rhinitis model sensitized by Artemisia annua pollen allergen, and explore the humoral and cellular immune indicators that can be used for the evaluation of allergic reactions. Methods:Using BALB/c mice as experimental animals, using Artemisia annua pollen allergen extract as sensitizing protein, through different content of the main allergen Art a1 and different sensitization times, different immunization programs were set to immunize mice subcutaneously, One week and five weeks after the last immunization, Artemisia annua pollen allergen extract containing 50 μg/ml and 500 μg/ml Art a1 was used for nasal stimulation, once a day, for 1 week each time.Observe the allergic reaction of mice, detect the pathological changes of nasal tissues, determine the levels and dynamic changes of antigen-specific IgE, IgG1, IgG2a and other antibodies in the serum of each group of mice. and detect the changes in the number of antigen-specific IL-4, IL-5, IL-2, IFN-γ and other lymphocytes in the spleen of mice. Results:Sensitized mice showed obvious scratching and sneezing reactions after being stimulated by antigen; obvious allergic inflammation appeared in nasal tissue; The increase in serum level of Artemisia annua pollen-specific IgE antibody was significantly correlated with the challenge antigen; The antigen-specific IL-4 lymphocytes in the spleen of the sensitized mice were significantly increased, but the IFN-γ-specific lymphocytes did not change significantly. Conclusions:The successful establishment of a mouse model of Artemisia annua pollen allergen allergy is the first domestic use of ELISPOT technology to detect an increase in the number of antigen-specific IL-4 lymphocytes in Artemisia annua allergy mice, laying a foundation for the subsequent evaluation of the efficacy of preparations for desensitization treatment basis.