Der p1 T细胞表位融合蛋白对哮喘小鼠的特异性免疫治疗效果

Specific immunotherapeutic effect of recombinant Der p1 T cell epitope fusion protein on asthma in a mouse model

目的探讨重组变应原Der p1 T融合蛋白对屋尘螨粗提液诱导的哮喘小鼠特异性免疫治疗的效果。方法将30只雌性BALB/c小鼠随机均分为3组,分别为对照组、哮喘组和Der p1 T融合蛋白免疫治疗组(SIT组)。哮喘组和SIT组小鼠分别在第0、7、14天给予腹腔注射屋尘螨粗提液200μl/鼠(蛋白含量10μg/200μl),对照组小鼠注射等量PBS。哮喘组、SIT组小鼠于第21天采用屋尘螨粗提液雾化激发(蛋白浓度为0.5μg/ml),30 min/d,连续7 d,观察并记录小鼠哮喘发作情况,对照组使用等量PBS雾化激发。SIT组小鼠于第21天雾化前0.5 h,腹腔注射Der p1 T融合蛋白(100μg/ml) 200μl进行特异性免疫治疗,连续7d,对照组、哮喘组注射等量PBS。各组小鼠于末次雾化激发后24 h内取眼球血,收集血清,气管插管收集肺泡灌洗液(BALF),ELISA检测血清中特异性IgE和IgG2a抗体水平及BALF中细胞因子γ干扰素(IFN-γ)、白细胞介素-4 (IL-4)、IL-10和IL-17A的含量;流式细胞仪检测脾组织中Th1/Th2和Th17/Treg细胞群落的改变;HE染色镜下观察小鼠肺组织病理形态。采用SPSS 18.0统计软件进行统计学分析。结果各组小鼠雾化激发后,对照组小鼠仅出现短暂的轻微烦躁症状,哮喘组小鼠表现出明显的烦躁不安、喘息、呼吸加快加深,SIT组小鼠经免疫治疗后,有轻微的喘息症状,症状较哮喘组小鼠有所改善。ELISA检测结果显示,哮喘组小鼠血清中特异性IgE抗体含量为(31.49±4.32) IU/ml,高于对照组的(8.53±1.92) IU/ml和SIT组的(16.68±2.45) IU/ml (P<0.01);哮喘组小鼠血清中抗原特异性IgG2a抗体含量为(19.56±3.89)μg/ml,低于对照组的(42.43±2.07)μg/ml和SIT组的(36.96±5.04)μg/ml (P <0.01)。哮喘组小鼠IFN-γ和IL-10水平分别为(134.23±22.49)和(22.43±8.27) pg/ml,低于对照组的(212.36±33.21)和(72.84±21.42) pg/ml (P<0.05、0.01);SIT组小鼠IFN-γ和IL-10水平分别为(183.76±24.66)和(61.05±7.97) pg/ml,与哮喘组相比,差异有统计学意义(P<0.05或0.01)。哮喘组小鼠IL-4和IL-17A水平分别为(165.45±34.59)和(464.21±41.36) pg/ml,高于对照组的(21.31±5.26)和(115.74±30.82)μg/ml (P<0.01);SIT组小鼠IL-4和IL-17A水平分别为(64.15±17.33)和(271.61±27.07) pg/ml,与哮喘组相比,差异有统计学意义(P<0.01)。流式细胞仪检测结果显示,哮喘组小鼠脾组织CD4^+T淋巴细胞中Th1和Treg细胞比例分别为2.8%和4.9%,较对照组的3.7%和10.3%显著降低(P<0.05、0.01);SIT组Th1和Treg细胞比例分别为3.1%和8.8%,较哮喘组小鼠的有所升高(P<0.05、0.01)。哮喘组Th2和Th17细胞比例分别为2.8%和2.2%,较对照组的1.0%和0.3%显著升高(P<0.01),SIT组Th2和Th17细胞比例分别为1.7%和0.6%,较哮喘组小鼠有所降低(P<0.01)。肺组织病理切片结果显示,哮喘组小鼠肺组织终末细支气管有明显炎性细胞浸润,支气管平滑肌纤维断裂,上皮细胞脱落;SIT组小鼠肺组织病理变化较哮喘组的有明显好转,支气管壁增厚减少,炎性细胞浸润情况得到改善。结论 Der p1T融合蛋白对哮喘小鼠的特异性免疫治疗有一定的效果。

Objective To explore the specific immunotherapeutic effect of Der p1 T cell epitope fusion protein on asthma in a mouse model.Methods Total 30 female BALB/c mice were randomly divided into three groups:control group,asthma group and specific immunotherapy group(SIT) treated with Der p1 T epitope fusion protein.The mice in asthma and SIT groups were sensitized with intraperitoneal injection of 200 μl(10 μg) crude extracts of house dust mite on day 0,7 and 14,while the mice in control group were given with intraperitoneal injection of the same volume of PBS solution.Then the mice in asthma and SIT groups were challenged by spray inhalation of house dust mite extracts(0.5 μLg/ml) for 30 minutes every day started from 21 st day.The asthmatic symptoms of the challenged mice were observed and recorded for 7 consecutive days.Control group was given with the same volume of PBS.The mice in SIT group were treated with intraperitoneal injection of 20 μg Der p1 T cell epitope fusion protein in total volume of 200 μl 30 min before each challenge for 7 consecutive days.Mice in control group and asthma group were injected intraperitoneally with the same volume of PBS.The blood was collected from each mouse and sera were collected 24 hours after the last challenge,mice were euthanized and bronchoalveolar lavage fluid was collected by trachael intubation.ELISA kit was used to detect the serum levels of antigen-specific IgE and IgG2 a and cytokines,including IL-10,IL-4,IFN-γ and IL-17 A in the bronchoalveolar lavage fluid.Flow cytometry analysis was used to determine the change of Thl/Th2 and Th17/Treg cells.Lung tissues were taken for histological observation of the pathological changes.Results After being sensitized and challenged,the mice in the control group only had a short period of mild restlessness.The mice in asthma group showed obvious symptoms of restlessness,wheezing and out of breath.After immunotherapy,the mice in the SIT group had reduced asthmatic symptoms,significant improvement than mice in asthma group without treatment.The results of ELISA showed that the amount of specific IgE antibody in asthma group was(31.49±4.32) IU/ml,which was significantly higher than that in control group [(8.53±1.92) IU/ml](P <0.01) and in SIT group [(16.68±2.45) IU/ml](P <0.01).The amount of antibody IgG2 a in asthma mice was(19.56±3.89) μg/ml,which was lower than that in control group [(42.43±2.07) μg/ml](P <0.01) and in SIT group [(36.96±5.04) μg/ml](P <0.01).The levels of IFN-γ and IL-10 in asthma group were(134.23±22.49) pg/ml,(22.43±8.27) pg/ml,respectively,which are significantly lower than those in control group [(212.36±33.21) and(72.84±21.42) pg/ml,respectively](P <0.05,0.01).However,after being treated with SIT,the levels of IFN-γ and IL-10 had significantly recovered compared to the asthma group without SIT treatment,as(183.76±24.66) pg/ml,(61.05±7.97) pg/ml,respectively(P <0.05,0.01).The levels of IL-4 and IL-17 A in asthma group were(165.45±34.59) pg/ml,(464.21±41.36)pg/ml,respectively,which are significantly higher than those in control group [(21.21±5.26) and(115.74±30.82)pg/ml,respectively](P <0.05,0.01).Treatment with SIT reduced IL-4 and IL-17 A levels to(64.15±17.33) pg/ml,(271.61±27.07) pg/ml,respectively,with significant difference compared to that in asthmat group without treatment(P <0.05,0.01).Consistently,the results of flow cytometry showed that the percentages of Thl and Treg cells in CD4^+T lymphocytes of spleen in asthma group were 2.8% and 4.9% respectively,which were significantly lower than those in control group(3.7% and 10.3%)(P <0.05,0.01).The treatment with SIT significantly stimulated Thl and Treg cells to 3.1% and 8.8%,respectively,with statistical significance compared to asthma group without treatment(P <0.05,0.01).The percentages of Th2 and Th17 cells in CD4^+T lymphocytes of spleen in asthma group were 2.8% and 2.2% respectively,which was significantly higher than those in control group(1.0% and0.3%)(P <0.01).The treatment of SIT reduced Th2 cells and Th17 cells to 1.7% and 0.6% respectively,with significant difference compared to asthma group without treatment(P <0.01).Pathological results showed that there was significant inflammatory cell infiltration around the bronchioles,broken muscle fiber and the collapsed epithelial cells were observed in asthma group.These pathological changes were much improved in mice with SIT treatment with reduced bronchus wall thickness and inflammatory cells infiltration.Conclusion Der p1 T epitope fusion protein has significant immunotherapeutic effect on asthma in a mouse model.