论文部分内容阅读
目的探讨以屋尘螨(Dermatophagoides pteronyssinus)1类变应原T细胞表位融合肽(TAT-Ih CDPTCE)为疫苗,评价其对过敏性哮喘小鼠特异性免疫治疗的效果。方法 120只SPF级BALB/c小鼠随机均分为PBS组(阴性对照,A组)、Pro Der p 1变应原致敏组(B组)、Pro Der p 1变应原免疫治疗组(C组)、DPTCE蛋白免疫治疗组(D组)、TAT-DPTCE蛋白免疫治疗组(E组)和TAT-Ih C-DPTCE蛋白免疫治疗组(F组),每组20只。分别于第0、7、14天,A组小鼠腹腔注射PBS,B~F组小鼠均腹腔注射屋尘螨变应原提取液10μg。第21天起,A组小鼠雾化吸入PBS,B~F组小鼠均吸入0.5μg/ml屋尘螨变应原提取液,1次/d×30 min,连续7 d。C~F组小鼠于第25~27天雾化前30 min分别腹腔注射100μg/ml Pro Der p 1、DPTCE、TAT-DPTCE和TAT-Ih CDPTCE溶液各200μl,进行特异性免疫治疗,A、B组小鼠分别注射200μl PBS。最后1次雾化后24 h,处死各组小鼠。HE染色观察小鼠肺组织病理变化。分别收集各组20只小鼠支气管肺泡灌洗液(BALF),ELISA检测BALF中γ干扰素(IFN-γ)、白细胞介素13(IL-13)、IL-10和β转化生长因子(TGF-β)的水平,并计数嗜酸粒细胞数量(EOS)。分别取各组5只小鼠眼眶血,ELISA检测血清中变应原特异性Ig E、Ig G_1和IgG_(2a)的抗体水平。结果 HE染色镜检结果显示,与B组比较,F组小鼠支气管周围嗜酸粒细胞增多、上皮细胞脱落和支气管上皮细胞肥大等肺部炎症明显减轻。小鼠BALF中,F组的IFN-γ水平为(298.75±26.09)pg/ml,高于B组(158.71±20.89)pg/ml、C组(210.38±18.92)pg/ml、D组(229.44±13.00)pg/ml和E组(233.24±20.39)pg/ml(P<0.01);IL-10和TGF-β水平与IFN-γ相似,F组IL-10和TGF-β水平分别为(105.32±7.24)和(119±9.33)pg/ml,均高于B组(23.29±3.18)和(41.19±4.63)pg/ml、C组(43.54±4.28)和(60.19±6.47)pg/ml、D组(51.33±6.19)和(69.34±8.27)pg/ml、E组(52.78±7.83)和(71.22±7.94)pg/ml(P<0.01);而C、D、E和F组的IL-13水平分别为(47.35±4.71)、(41.90±4.28)、(41.05±6.50)和(18.53±5.67)pg/ml,均低于B组(66.68±6.63)pg/ml(P<0.01),其中F组IL-13水平最低。B组小鼠BALF中的EOS数量为(5.65±0.91)×10~5/ml,高于A组(0.45±0.39)×10~5/ml(P<0.01),而C、D、E和F组的嗜酸粒细胞数量均显著下降,分别为(4.00±0.59)×10~5/ml、(3.39±0.63)×10~5/ml、(3.24±0.69)×10~5/ml和(1.42±0.49)×10~5/ml(P<0.01)。血清中抗体水平的ELISA检测结果显示,F组小鼠血清Ig E水平为(5.26±1.72)ng/ml,低于B组(32.81±2.98)ng/ml、C组(20.06±3.17)ng/ml、D组(17.06±3.18)ng/ml和E组(16.23±3.61)ng/ml(P<0.01);F组中血清Ig G_1水平为(9.85±1.42)ng/ml,亦低于B组(43.72±3.05)ng/ml、C组(31.54±4.25)ng/ml、D组(25.20±2.91)ng/ml和E组(23.96±4.12)ng/ml(P<0.01或P<0.05);F组中血清Ig G_2a水平为(43.10±1.34)ng/ml,高于B组(12.61±1.87)ng/ml、C组(23.37±2.67)ng/ml、D组(25.60±2.10)ng/ml和E组(25.91±1.33)ng/ml(P<0.01)。结论通过TAT-Ih C-DPTCE免疫治疗小鼠哮喘,可有效改善小鼠变态反应性气道及肺部炎症。
Objective To investigate the effect of specific immunotherapy on allergic asthma in mice by using TAT-Ih CDPTCE as a vaccine against Dermatophagoides pteronyssinus. Methods 120 SPF BALB / c mice were randomly divided into PBS group (negative control, group A), Pro Der p 1 allergen sensitized group (group B), Pro Der p 1 allergen immunotherapy group Group C), DPTCE protein immunotherapy group (group D), TAT-DPTCE protein immunotherapy group (group E) and TAT-Ih C-DPTCE protein immunotherapy group (group F). Groups A and B were intraperitoneally injected with PBS on days 0, 7 and 14, and mice in groups B to F were intraperitoneally injected with 10 μg of house dust mite allergen extract. On day 21, group A mice were inhaled into PBS. Group B and F mice inhaled 0.5μg / ml house dust mite allergen extract once a day for 7 days. The mice in group C ~ F were intraperitoneally injected with 200μg of Pro Der p 1, DPTCE, TAT-DPTCE and TAT-Ih CDPTCE respectively intraperitoneally 30 min prior to the first 25 to 27 days of nebulization. Group B mice were injected with 200μl PBS. 24 hours after the last atomization, all mice were sacrificed. The histopathological changes of lung in mice were observed by HE staining. Bronchoalveolar lavage fluid (BALF) was collected from 20 mice in each group. The levels of IFN-γ, IL-13, IL-10 and TGFβ in BALF -β), and count the number of eosinophils (EOS). Orbital blood was collected from 5 mice in each group, and the serum levels of allergen-specific Ig E, Ig G 1 and IgG 2 a were detected by ELISA. Results The results of HE staining showed that compared with group B, the inflammation in lungs of bronchial epithelium such as eosinophilia, epithelial cells shedding and bronchial epithelial cell hypertrophy in group F were significantly reduced. The level of IFN-γ in group F was (298.75 ± 26.09) pg / ml in BALF of mice, which was higher than that of group B (158.71 ± 20.89) pg / ml and group C (210.38 ± 18.92) pg / ± 13.00) pg / ml and E group (233.24 ± 20.39) pg / ml (P <0.01). The levels of IL-10 and TGF-β were similar to those of IFN-γ and the levels of IL-10 and TGF- (23.39 ± 3.18) and (41.19 ± 4.63) pg / ml in group B, (43.54 ± 4.28) and (60.19 ± 6.47) pg / ml in group C (51.33 ± 6.19) and (69.34 ± 8.27) pg / ml in group D, 52.78 ± 7.83 and 71.22 ± 7.94 pg / ml in group E (P <0.01) The levels of IL-13 were (47.35 ± 4.71), (41.90 ± 4.28), (41.05 ± 6.50) and (18.53 ± 5.67) pg / ml, ), Among which the lowest level of IL-13 in F group. The number of EOS in BALF in group B was (5.65 ± 0.91) × 10-5 / ml, higher than that in group A (0.45 ± 0.39) × 10-5 / ml (P <0.01) (4.00 ± 0.59) × 10-5 / ml, (3.39 ± 0.63) × 10-5 / ml and (3.24 ± 0.69) × 10-5 / ml in group F, and the number of eosinophils in group F were significantly decreased (1.42 ± 0.49) × 10 ~ 5 / ml (P <0.01). The level of serum IgE in serum of group F was (5.26 ± 1.72) ng / ml, lower than that of group B (32.81 ± 2.98) ng / ml and group C (20.06 ± 3.17) ng / ml, 17.06 ± 3.18 ng / ml in D group and 16.23 ± 3.61 ng / ml in E group (P <0.01). The serum Ig G_1 level in group F was (9.85 ± 1.42) ng / ml, (43.72 ± 3.05) ng / ml, C (31.54 ± 4.25) ng / ml, D (25.20 ± 2.91) ng / ml and E (23.96 ± 4.12) ng / ml ), The level of serum Ig G_2a in group F was (43.10 ± 1.34) ng / ml, higher than that in group B (12.61 ± 1.87) ng / ml and group C (23.37 ± 2.67) ng / ng / ml and E group (25.91 ± 1.33) ng / ml (P <0.01). Conclusion Immunotherapy of asthma in mice by TAT-Ih C-DPTCE can effectively improve allergic airway and lung inflammation in mice.