重组蛋白Blo t 21 T特异性免疫治疗哮喘小鼠效果研究

doi:10.12140/j.issn.1000-7423.2019.03.008

摘要/Abstract

摘要：

目的探讨热带无爪螨重组蛋白Blo t 21 T特异性免疫治疗哮喘小鼠的效果。方法将30只BALB/c雌性小鼠随机分为对照组、哮喘组和免疫治疗组3组,每组10只。哮喘组和免疫治疗组小鼠分别于第0、7、14天腹腔注射200 μl热带无爪螨变应原粗提液[含蛋白10 μg,Al(OH)₃ 2 mg],第21天开始,在每天同一时间用热带无爪螨变应原粗提液(蛋白浓度为0.5 μg/ml)雾化激发小鼠,连续雾化7 d,每次30 min。免疫治疗组在第25~27天雾化激发前30 min,腹腔注射重组蛋白Blo t 21 T 200 μl（含蛋白10 μg）。对照组用PBS替代。最后一次雾化24 h后,水合氯醛麻醉小鼠,眼球采血并收集肺泡灌洗液（BALF）和肺组织。刘氏染色法计数BALF中嗜酸粒细胞,ELISA检测BALF中细胞因子白细胞介素4（IL-4）、IL-13、IL-17和γ干扰素（IFN-γ）水平,以及血清中的IgE和IgG_2a抗体水平。制作肺组织病理切片,HE染色观察。多组数据的统计分析采用单因素F检验,两组数据的统计分析采用SNK检验。结果免疫治疗组小鼠BALF中嗜酸粒细胞数量为（1.23 ± 0.35）×10⁵/ml,低于哮喘组的（5.32 ± 1.22）×10⁵/ml（P < 0.05）;3组小鼠BALF中嗜酸粒细胞数量差异有统计学意义（P < 0.05）。 ELISA检测结果显示,免疫治疗组小鼠BALF中IL-4、IL-13和IL-17水平分别为（70.96 ± 17.62）、（159.20 ± 21.08）和（220.80 ± 25.95）pg/ml,低于哮喘组的（111.96 ± 13.62）、（321.40 ± 31.88）和（425.40 ± 34.83）pg/ml（P < 0.05）,高于对照组的（12.57 ± 3.62）、（109.62 ± 15.38）和（114.38 ± 18.43）pg/ml（P < 0.05）;免疫治疗组IFN-γ水平为（316.80 ± 29.12）pg/ml,高于哮喘组和对照组的（72.84 ± 10.10）、（125.60 ± 22.38）pg/ml（P < 0.05）。免疫治疗组小鼠血清中IgG_2a、IgE抗体水平分别为（49.73 ± 10.95）、（316.80 ± 29.12）μg/ml,高于哮喘组的（27.30 ± 4.83）、（57.40 ± 15.96）μg/ml（P < 0.05）。肺组织HE染色观察显示,哮喘组可见较明显的气管壁增厚,血管黏膜和肺组织周围有较多的炎症细胞浸润;免疫治疗组与哮喘组相比,上述症状减轻,支气管壁增厚减少,炎症细胞和分泌物均较少。结论热带无爪螨重组蛋白Blo t 21 T特异性免疫治疗可以减轻哮喘小鼠症状。

关键词: 免疫治疗, BALB/c小鼠, 哮喘, 热带无爪螨, 重组蛋白

Abstract:

Objective To investigate the effect of allergen-specific immunotherapy of recombinant protein Blo t 21 T on asthma in mice sensitized by the extracts of mite Blomia tropicalis. Methods Thirty mice were randomly divided into three groups: the asthma group, the immunotherapy group, and the control group (PBS group)(n = 10 in each group). Mice in the asthma and immunotherapy group were sensitized by intraperitoneal injection of 10 μg B. tropicalis crude extracts formulated with 2 mg Al(OH)₃ in total volume of 200 μl on day 0, 7 and 14. On the 21^th day, the mice were challenged with the same allergen extracts (0.5 μg/ml) through spray inhalation for 30 min for consecutive 7 days. In the immunotherapy group, mice were treated intraperitoneally with 10 μg recombinant Blo t 21 T protein 30 min before the challenge inhalation from 25^th to 27^th days. In the asthma group, mice were not treated. In the control group, mice were not sensitized and treated, only receiving PBS. All mice were sacrificed 24 h after the last inhalation, the sera, bronchoalveolar lavage fluid (BALF) and the lung tissue were collected. The number of eosinophils (EOS) was counted in BALF, the levels of cytokine IL-4, IL-13, IL-17 and IFN-γ in BALF, IgE and IgG_2a in sera were determined by ELISA. The lung histochemical sections were made to evaluate the pathological changes. Single factor F test was used for multiple group comparison. SNK test was used to compare the data from two groups. Results The count of EOS in immunotherapy group was (1.23 ± 0.35) × 10⁵/ml, which was significantly lower than that in asthma group [(5.32 ± 1.22) × 10⁵/ml] (P < 0.05). The levels of IFN-γ, IL-4, IL-13, IL-17 in the immunotherapy group [(316.80 ± 29.12), (70.96 ± 17.62), (159.20 ± 21.08) and (220.80 ± 25.95) pg/ml, respectively] was significantly lower than that in the asthma group [(72.84 ± 10.10), (111.96 ± 13.62), (321.40 ± 31.88) and (425.40 ± 34.83) pg/ml, respectively] (P < 0.05). The levels of IFN-γ, IL-4, IL-13 and IL-17 in asthma group was significantly different from the levels in control group [(125.60 ± 22.38), (12.57 ± 3.62), (109.62 ± 15.38) and (114.38 ± 18.43) pg/ml] (P < 0.05). The level of serological IgG_2a and IgE in the immunotherapy group [(49.73 ± 10.95) and (316.80 ± 29.12) μg/ml, respectively] was higher than that in the asthma group [(27.30 ± 4.83) and (57.40 ± 15.96) μg/ml, respectively] (P < 0.05), however, the levels of IgE in asthma group is higher than that in control group(P < 0.05). HE staining of lung tissue showed that lung tissue in immunotherapy group had reduced pathological changes characterized by the reduced thickened tracheal wall and less inflammatory cell infiltration around the vascular mucosa and lung tissue compared with the un-treated asthma group. Conclusion the Blomia tropicalis recombinant protein Blo t 21 T has allergen-specific immunotherapeutic effect on mite-induced asthma in a mice model.

Key words: Immunotherapy, BALB/c mice, Asthma, Blomia tropicalis, Reconbinant proten

中图分类号:

R384.429

柴强, 李朝品. 重组蛋白Blo t 21 T特异性免疫治疗哮喘小鼠效果研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(3): 286-290.

Qiang CHAI, Chao-pin LI. Allergen-specific immunotherapeutic effect of recombinant Blo t 21 T protein on asthma in a mouse model[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(3): 286-290.

图/表 2

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