中国寄生虫学与寄生虫病杂志

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屋尘螨1类变应原T细胞表位融合肽对过敏性哮喘小鼠的免疫治疗效果

李朝品*,赵蓓蓓,湛孝东   

  1. 皖南医学院医学寄生虫学教研室,芜湖 241002
  • 出版日期:2016-06-30 发布日期:2016-10-28

Immunotherapeutic Effect of Dermatophagoides pteronyssinus Group 1 Allergen T Cell Epitope Peptide Against Allergic Asthma in Mice

LI Chao-pin*, ZHAO Bei-bei, ZHAN Xiao-dong   

  1. Department of Medical Parasitology,Wannan Medical College, Wuhu 241002, China
  • Online:2016-06-30 Published:2016-10-28

摘要: 目的 探讨以屋尘螨(Dermatophagoides pteronyssinus)1类变应原T细胞表位融合肽(TAT-IhC-DPTCE)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)、ProDer p 1变应原致敏组(B组)、ProDer 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-IhC-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10 μg。第21天起,A组小鼠雾化吸入PBS,B~F组小鼠均吸入0.5 μg/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100 μg/ml ProDer p 1、DPTCE、TAT-DPTCE和TAT-IhC-DPTCE溶液各200 μl,进行特异性免疫治疗,A、B组小鼠分别注射200 μl 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IgE、IgG1和IgG2a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)×105/ml,高于A组(0.45±0.39)×105/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)×105/ml、(3.39±0.63)×105/ml、(3.24±0.69)×105/ml和(1.42±0.49)×105/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IgE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IgG1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IgG2a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-IhC-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。

关键词: 屋尘螨, 变应原, 特异性免疫治疗, T细胞表位

Abstract: Objective To study the specific immunotherapeutic effect of Dermatophagoides pteronyssinus group 1 major allergen T-cell fusion epitope peptide vaccine TAT-IhC-DPTCE against allergic asthma. Methods One hundred and twenty SPF-grade BALB/c mice were randomized into PBS group(group A), asthma group (group B), and immune treatment groups respectively receiving intraperitoneal(i.p.) injections of ProDer p 1 allergen(group C), DPTCE (group D), TAT-DPTCE(group E) or TAT-IhC-DPTCE(group F) (n=20 in each group). In detail, PBS (group A) or allergen extract derived from Dermatophagoides pteronyssinus (groups B-F, 10 μg) was intraperitoneally injected on days 0, 7 and 14, and was continued by aerosol inhalation from day 21 for 7 consecutive days (0.5 μg/ml, once/day, 30 min each time). The mice in groups C-F received i.p. injections of 100 μg/ml ProDer p 1, DPTCE, TAT-DPTCE and TAT-IhC-DPTCE respectively 30 min prior to inhalation challenge on days 25-27 as a specific immunotherapy, while those in groups A and B received 200 μl PBS. Twenty-four hours after the last inhalation challenge, all the mice were sacrificed. The lung histopathological changes were examined by HE staining. The levels of IFN-γ, IL-13, IL-10 and TGF-β in the bronchoalveolar lavage fluid(BALF) was determined with ELISA, and eosinophils in the BALF were counted(n=20 mice in each group). The serum level of IgE, IgG1 and IgG2a in orbital blood was determined by ELISA(n=5 mice in each group). Results HE staining revealed increased BALF eosinophils and decreased pulmonary inflammation in group F compared with group B. The IFN-γ level in group F [(298.75±26.09) pg/ml] was significantly higher than those in groups B[(158.71±20.89) pg/ml], C[(210.38±18.92) pg/ml], D [(229.44±13.00) pg/ml] and E[(233.24±20.39) pg/ml] (all P<0.01). Similar results were also found for IL-10 and TGF-β, while the IL-13 levels in groups C [(47.35±4.71) pg/ml], D [(41.90±4.28) pg/ml], E[(41.05±6.50) pg/ml] and F[(18.53±5.67) pg/ml] were all significantly lower than that in group B [(66.68±6.63) pg/ml](all P<0.01). The number of BALF eosinophils in group B [5.65±0.91]×105/ml] was significantly higher than that in group A [(0.45±0.39)×105/ml] (P<0.01), while the BALF eosinophils in groups C [(4.00±0.59)×105/ml], D [(3.39±0.63)×105/ml], E [(3.24±0.69)×105/ml] and F [(1.42±0.49)×105/ml] decreased after immune treatment(all P<0.01). ELISA results showed that the serum IgE level in group F [(5.26±1.72) ng/ml] was significantly lower than those in group B [(32.81±2.98) ng/ml] and the other 3 treatment groups[group C, (20.06±3.17) ng/ml; D, (17.06±3.18) ng/ml; E, (16.23±3.61) ng/ml]. Similar results were also obtained for IgG1. In contrast, the serum IgG2a level in group F[(43.10±1.34) ng/ml] was significantly higher than those in group B[(12.61±1.87) ng/ml] and the other 3 treatment groups [group C, (23.37±2.67) ng/ml; D, (25.60±2.10) ng/ml; E, (25.91±1.33) ng/ml] (all P<0.01). Conclusion Immunotherapy with chimeric TAT-IhC-DPTCE can effectively ameliorate the allergic airway response and pulmonary inflammation in mice.

Key words: Dermatophagoides pteronyssinus, Allergen, Specific immunotherapy, T-cell epitope