Abstract: Objective Objective To investigate the enzymatic hydrolysates of ProDer f 1and their hydrolytic products for specific im? munotherapy. Methods Methods The asthma models of mice made by ProDer f 1 allergen were treated by using two kinds of hydroly? sates as vaccine for analyzing their effects of immunotherapy. Fifty female BALB/c mice were randomly divided into 5 groups（n = 10 for each），i.e.，a PBS group，an asthma group，an immunotherapy group by ProDer f 1 protein，an immunotherapy groups by papain hydrolysates and trypsin hydrolysates. On day 0，7 and 14，the mice were intraperitoneally injected with 10 μg of ProDer f 1 allergen，which was dissolved in 100 μl PBS containing 2%（W/V）Al（OH） 3 suspension. At day 21，the animals were caged in the airway challenge apparatus，and challenged by nebulized inhalation of allergen suspension（0.5 μg/ml）for 30 min for 7 successive days. The mice were undergone allergen specific immunotherapy（ASIT）by intraperitoneal injection and of the immunotherapy group by ProDer f 1 protein，immunotherapy groups by papain hydrolysates and trypsin hydrolysates（100 μg/ml）in a dose of 200 μl of reactive allergens，30 min prior to the inhalation treatment at day 25，26 and 27，respectively. The PBS group was managed with both intraperitoneal injection and aerosol of PBS. Twenty?four hours after the last challenge，all the mice were sacrificed. The bronchoalveolar lavage fluid（BALF）and sera were collected，and the splenocytes were cultured. The levels of IL ?4，IL?10，IL?17 and IFN?γ in BALF and supernatant of splenocytes cultured（SSCC）were detected by ELISA，and the serum levels of specific IgE and IgG2a antibodies were also detected by ELISA. Results Results Compared with the asthma group，the histo? logic examination of the lungs taken from the mice in the immunotherapy group by ProDer f 1 protein，immunotherapy groups by papain hydrolysates and trypsin hydrolysates showed alleviated peribronchial and perivascular inflammatory infiltration，absent? ly of eosinophils. The normal lung architectures were also exhibited，particularly，the epithelium was of normal size and morphol? ogy，similar to that of the PBS?challenged group. The levels of IL?4 in BLAF of the ASIT groups and asthma group were（231.61± 11.73）， （206.20±14.33）， （200.44±9.34）， （299.68±12.46）pg/ml；the levels of IL? 10 in BLAF were（361.87±13.62）， （376.27±20.57）， （413.57±12.98）， （171.28±19.79）pg/ml；the levels of IL?17 in BLAF were（142.12±5.01）， （128.27± 5.34）， （130.79 ± 6.30）， （273.59 ± 11.56）pg/ml；the levels of IFN? γ in BLAF were（229.60 ± 11.32）， （269.13 ± 11.98）， （282.25±19.65）， （147.76±11.36）pg/ml. The levels of IL? 4 in SCCS of the ASIT groups and asthma group were（218.54± 12.62）， （220.21±10.73）， （201.59±18.54）， （256.86±15.53）pg/ml；the levels of IL?10 were（360.45±13.10）， （383.41± 19.81）， （413.51±13.14）， （173.50±20.25）pg/ml；the levels of IL?17 were（154.23±5.18）， （137.72±6.66）， （141.01±7.35）， （297.55±8.97）pg/ml，the levels of IFN?γ were（243.22±25.01）， （275.20±14.65）， （284.67±25.87）， （154.54±17.45）pg/ml. The levels of antigen? specific IgE antibody of the ASIT groups and asthma group were（309.66±13.56）， （256.61±40.64）， （248.83±10.51）， （359.60±29.48）μg/ml，and the antigen? specific IgG2a antibody levels were（8.87±0.82）， （9.15±0.83）， （10.56±1.68）， （7.04±0.42）μg/ml. The resulting serum antigen?specific IgE antibody levels suggested that the IgE levels in the immunotherapy group by ProDer f 1 protein，immunotherapy groups by papain hydrolysates and trypsin hydrolysates were signifi? cantly lower than that in the asthma group. Conversely，the level of antigen?specific IgG2a in sera was significantly higher in the immunotherapy group by ProDer f 1 protein，immunotherapy groups by papain hydrolysates and trypsin hydrolysates than that in the asthmatic group. The levels of IL?4，IL?17 in BALF and SCCS in the immunotherapy group by ProDer f 1 protein，immuno? therapy groups by papain hydrolysates and trypsin hydrolysates significantly decreased，compared with that in the asthma group. However，the levels of IL?10 and IFN?γ in BALF and SCCS in the immunotherapy group by ProDer f 1 protein，immunotherapy groups by papain hydrolysates and trypsin hydrolysates were increased dramatically in the immunotherapy group by ProDer f 1 protein，immunotherapy groups by papain hydrolysates and trypsin hydrolysates than that in the asthma group. Conclusion Conclusion The hydrolytic products above?mentioned can alleviate asthmatic symptoms effectively after the antigen?specific immunotherapy in murine asthma models.

Key words: Dermatophagoides farinae；ProDer f1 protein；Hydrolysis；Asthma；Immunotherapy

摘要： 目的 目的 探讨粉尘螨1类变应原 （ProDer 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 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、 ProDer 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ProDe 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ProDe 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IgE、 IgG2a水平。结果 结果 各免疫治疗组对ProDer 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ProDer 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。ProDer 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IgE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， IgG2a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IgE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IgG2a、 BALF和脾细胞培养上 清中IFN?γ、 IL?10含量均比哮喘组高。结论 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ProDer f 1蛋白致敏的小鼠变态反应性气道及肺部炎症。

关键词: 粉尘螨； ProDer f 1蛋白； 水解； 哮喘； 免疫治疗