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目的探讨粉尘螨1类变应原(Pro Der f 1)两种酶解产物(木瓜蛋白酶水解产物、胰蛋白酶水解产物)对过敏性哮喘特异性免疫治疗的效果。方法 50只6~8周龄的SPF雌性小鼠随机分为磷酸盐缓冲液(PBS)组、哮喘组、Pro Der f 1蛋白治疗组、木瓜蛋白酶水解产物免疫治疗组和胰蛋白酶水解产物免疫治疗组5组,每组各10只。用纯化的Pro De rf1蛋白于第0、7、14天腹腔注射致敏小鼠,第21天开始雾化吸入激发、连续7 d。各免疫治疗组于第25~27天雾化前30 min分别用Pro De rf1蛋白和其酶解产物进行特异性免疫治疗,PBS组用PBS进行腹腔注射和雾化激发。最后一次雾化激发后24 h内,各组小鼠引颈处死。观察各组小鼠肺组织病理变化,ELISA测定支气管肺泡灌洗液(BALF)和脾细胞培养上清中IL-4、IL-10、IL-17和IFN-γ含量及血清特异性抗体Ig E、Ig G2a水平。结果各免疫治疗组对Pro Der f 1哮喘小鼠进行免疫治疗后,肺组织切片观察表明:酶解产物治疗组和Pro Der f 1蛋白治疗组变态反应性炎症较哮喘组显著减轻,且炎性细胞浸润较哮喘组也显著减少,气道上皮结构与PBS组相近。Pro Der f 1蛋白治疗组、木瓜蛋白酶水解产物免疫治疗组、胰蛋白酶水解产物免疫治疗组和哮喘组小鼠BALF中的IL-4含量分别为(231.61±11.73)、(206.20±14.33)、(200.44±9.34)、(299.68±12.46)pg/ml,IL-10含量分别为(361.87±13.62)、(376.27±20.57)、(413.57±12.98)、(171.28±19.79)pg/ml,IL-17含量为(142.12±5.01)、(128.27±5.34)、(130.79±6.30)、(273.59±11.56)pg/ml,IFN-γ含量为(229.60±11.32)、(269.13±11.98)、(282.25±19.65)、(147.76±11.36)pg/ml;各治疗组和哮喘组小鼠脾细胞分泌因子IL-4含量分别为(218.54±12.62)、(220.21±10.73)、(201.59±18.54)、(256.86±15.53)pg/ml,IL-10含量分别为(360.45±13.10)、(383.41±19.81)、(413.51±13.14)、(173.50±20.25)pg/ml,IL-17含量为(154.23±5.18)、(137.72±6.66)、(141.01±7.35)、(297.55±8.97)pg/ml,IFN-γ含量为(243.22±25.01)、(275.20±14.65)、(284.67±25.87)、(154.54±17.45)pg/ml;各治疗组和哮喘组小鼠血清中Ig E抗体含量分别为(309.66±13.56)、(256.61±40.64)、(248.83±10.51)、(359.60±29.48)μg/ml,Ig G2a抗体含量分别为(8.87±0.82)、(9.15±0.83)、(10.56±1.68)、(7.04±0.42)μg/ml。各免疫治疗组血清抗原特异性Ig E抗体、BALF和脾细胞分泌的IL-4、IL-17均比哮喘组低;血清抗原特异性抗体Ig G2a、BALF和脾细胞培养上清中IFN-γ、IL-10含量均比哮喘组高。结论应用上述两种蛋白酶水解产物对小鼠哮喘模型进行特异性免疫治疗,可有效改善由Pro Der f 1蛋白致敏的小鼠变态反应性气道及肺部炎症。
Objective To investigate the effect of two kinds of enzymatic hydrolyzate (papain hydrolyzate and trypsin hydrolyzate) of Pro Der f 1 on the specific immunotherapy of allergic asthma. Methods Fifty SPF female mice aged 6-8 weeks were randomly divided into phosphate buffered saline (PBS) group, asthma group, Pro Der f 1 protein treatment group, papain hydrolyzate immunotherapy group and trypsin hydrolyzate immunotherapy Group 5, each group of 10. Sensitized mice were injected intraperitoneally with purified Pro De rf1 protein on days 0, 7 and 14, and inhalation challenge was started on day 21 for 7 days. Each immunotherapy group was immunized with Pro De rf1 protein and its enzymatic products for 30 min before fogging on days 25-27, respectively. The PBS group was injected intraperitoneally with PBS and atomized. Within 24 h after the last atomization challenge, mice in each group were euthanized. The pathological changes of lung tissue in each group were observed. The levels of IL-4, IL-10, IL-17 and IFN-γ in bronchoalveolar lavage fluid (BALF) and spleen cell culture supernatant were detected by ELISA and serum IgE , Ig G2a levels. Results Immunohistochemical results of the lungs of Pro Der f 1 asthmatic mice in each immunotherapy group showed that the allergic inflammation in the treated group and the Pro Der f 1 protein group was significantly reduced compared with the asthma group and the inflammatory Cell infiltration is also significantly reduced compared with asthma group, airway epithelium structure and PBS group similar. The levels of IL-4 in BALF of mice treated with Pro Der f 1 protein, papain hydrolyzate immunotherapy, trypsin hydrolyzate immunotherapy and asthma were (231.61 ± 11.73), (206.20 ± 14.33) and 200.44 ± 9.34), (299.68 ± 12.46) pg / ml and IL-10 contents were 361.87 ± 13.62, 376.27 ± 20.57, 413.57 ± 12.98, 171.28 ± 19.79 pg / ml, IL- The content of IFN-γ was (142.12 ± 5.01), (128.27 ± 5.34), (130.79 ± 6.30), (273.59 ± 11.56) pg / ml and the content of IFN-γ was (229.60 ± 11.32), (269.13 ± 11.98) ) And (147.76 ± 11.36) pg / ml, respectively. The levels of IL-4 in spleen cells of mice in each treatment group and asthma group were (218.54 ± 12.62), (220.21 ± 10.73), (201.59 ± 18.54) and 15.53) pg / ml and IL-10 were (360.45 ± 13.10), (383.41 ± 19.81), (413.51 ± 13.14), (173.50 ± 20.25) pg / ml and IL- (137.72 ± 6.66), (141.01 ± 7.35) and (297.55 ± 8.97) pg / ml, IFN-γcontent was (243.22 ± 25.01), (275.20 ± 14.65), (284.67 ± 25.87) and (154.54 ± 17.45) pg / ml; The serum IgE antibody levels of the mice in each treatment group and the asthma group were (309.66 ± 13.56) The antibody levels of Ig G2a were (256.61 ± 40.64), (248.83 ± 10.51) and (359.60 ± 29.48) μg / ml respectively, and the antibody levels of Ig G2a were (8.87 ± 0.82), (9.15 ± 0.83), (10.56 ± 1.68) and (7.04 ± 0.42) μg / ml. IL-4 and IL-17 secreted by BALF and spleen cells were lower in each immunotherapy group than those in asthma group. Serum antigen-specific antibody Ig G2a, BALF and IFN-γ in the culture supernatant of spleen cells , IL-10 levels were higher than the asthma group. Conclusion Specific immunotherapy with the two protease hydrolysates on mouse asthma model can effectively improve allergic airway and lung inflammation induced by Pro Der f 1 protein in mice.