粉尘螨Ⅲ类重组变应原致敏的小鼠哮喘模型致敏效果分析

Allergenic effect of recombinant group 3 allergen from Dermatophagoides farinae

目的探讨粉尘螨Ⅲ类重组变应原(Der f 3)诱导的小鼠哮喘模型的致敏效果。方法 30只6～8周龄雌性SPF级BALB/c小鼠随机分为PBS组(阴性对照组)、卵清蛋白(OVA)组(阳性对照组)和Der f 3组(实验组),OVA组和Der f 3组分别使用OVA和纯化Der f 3蛋白于第0、7、14d进行腹腔注射致敏,于第21d开始雾化吸入,连续7d,PBS组则换成PBS进行腹腔注射和雾化吸入。最后一次雾化吸入24h后,分别观察肺组织病理变化、支气管肺泡灌洗液(BLAF)中的白细胞计数,酶联免疫吸附试验(ELISA)测定BLAF和脾细胞培养上清中IL-4、IL-2、IL-17和IFN-γ的含量及血清中IgG1、IgE抗体水平变化。结果 OVA组和Der f 3组小鼠肺部支气管、血管粘膜下及周围肺组织有明显的炎性细胞浸润、支气管上皮细胞部分断裂及脱落,血管壁明显水肿等,而PBS组未见明显病理改变;Der f 3组的BALF中白细胞总数[(19.99±1.03)×106个/mL]及嗜酸性粒细胞(EOS)[(1.81±0.07)×106个/mL]计数均明显高于PBS组(P<0.01);与PBS组相比:BALF中IL-4[(80.99±9.06)pg/mL]、IL-17[(209.69±31.86)pg/mL]呈高水平表达(P<0.01),而IL-2[(8.29±1.27)pg/mL]和IFN-γ[(55.04±18.85)pg/mL]含量则显著下降(P<0.01);上述指标在脾细胞培养上清中出现类似的变化。Der f 3组血清中IgE[(31.92±2.68)U/mL]、IgG1[(16.46±4.32)μg/mL]的含量表现为Th2型反应增强趋势;但OVA组和Der f 3组间所有指标差异均无统计学意义(P>0.05)。结论粉尘螨重组Der f 3蛋白可成功建立小鼠变态反应性气道及肺部炎症动物模型。

The aim of this study is to analyze the sensitized effect of murine asthma model induced by recombinant Der f 3 allergen from Dermatophagoides farinae.Thirty female BALB/c mice were randomly divided into 3 groups: PBS group(negative controls),ovalbumin(OVA) group(positive controls) and Der f 3 group(experimental group).OVA and Der f 3 groups were initially immunized intraperitoneally injection three times with OVA and purified Der f 3 protein on 0,7 and 14 d respectively.Subsequently,OVA and Der f 3 solution was inhaled for 30 min/d from day 21 to 27.The PBS group was injected and inhaled with PBS solution respectively at the same time after 24 h of the last time of inhalation.Differential cells in Bronchoalveolar Lavage Fluid(BALF) were counted and bronchopulmonary tissues were observed histopathologically.IL-2,IL-4,IL-17 and IFN-γ in BALF and supernatants of spleen cells,allergen-specific IgG1 and IgE in serum were also measured by ELISA.The results showed that in the OVA and Der f 3 groups,eosinophils infiltrated into bronchial mucosa,blood vessels and the surrounding lung tissue.The bronchial epithelium cells fell off,and the hema wall swelled obviously.No significant pathological change has been seen in the PBS group.The number of total cells [(19.99±1.03)×106/mL] and eosinophils [(1.81±0.07)×106/mL] in BALF were significantly different in PBS and Der f 3 groups(P<0.01).At the same time,the levels of IL-4 [(80.99±9.06) pg/mL] and IL-17 [(209.69±31.86) pg/mL] in BALF from OVA and Der f 3 groups were increased obviously(P<0.01),comparing with that in PBS group.On the contrary,levels of IL-2 [(8.29±1.27) pg/mL] and IFN-γ [(55.04±18.85) pg/mL] in BALF were reduced significantly(P<0.01).The same trends of cytokines above were also detected in the supernatants of spleen cell cultures.Moreover,the levels of IgE [(31.92±2.68) U/mL] and IgG1 [(16.46±4.32) g/mL] in OVA and Der f 3 groups were also significantly increased in serum,comparing with those in PBS group(P<0.01).The phenomenon indicated that Th2-type immune response was enhanced and Th1-type immune response was reduced,and no statistically significant difference was occurred between OVA and Der f 3 groups(P>0.05).In conclusion,the murine asthma model of allergic airway and lung inflammation induced by recombinant Der f 3 has been established successfully.