改良型甲苯二异氰酸酯哮喘小鼠模型的建立

目的采用气管滴注方式进行甲苯二异氰酸酯(toluene-diisocyanate,TDI)激发给药,改进TDI哮喘小鼠模型建立方法,并对模型的建立进行评价。方法将20只SPF级健康雌性BALB/c小鼠随机分为TDI组和溶剂对照组,每组10只。TDI组小鼠第1、8天于耳背分别涂抹20μl 0.3%TDI致敏,第15天采用气管滴注法给予20μl 0.01%TDI进行激发,溶剂对照组给予等量溶剂(丙酮与橄榄油混合物)。激发结束后,观察各组小鼠的行为学改变。致敏实验后每组取6只小鼠收集支气管肺泡灌洗液(BALF)并进行炎细胞计数分类,以ELISA法检测BALF上清中白细胞介素-4(IL-4)、γ-干扰素(IFN-γ)含量;以HE染色观察小鼠肺组织病理变化。结果激发结束后,TDI组小鼠均出现呼吸急促、点头呼吸、弓背、前肢缩抬等行为学改变,溶剂对照组未出现上述症状。TDI组小鼠BALF中白细胞总数、嗜中性粒细胞、淋巴细胞、单核细胞、嗜酸性粒细胞、嗜碱性粒细胞数目均高于溶剂对照组,且嗜中性粒细胞、嗜酸性粒细胞百分比高于溶剂对照组,BALF中细胞因子IL-4、IFN-γ含量高于溶剂对照组,差异均有统计学意义(P0.05)。HE染色可见TDI组小鼠肺组织内支气管管壁破坏明显,管腔增厚,支气管管腔内及肺泡腔内有大量的炎细胞浸润,而溶剂对照组未见明显病理变化。结论采用气管滴注方式进行TDI激发给药,可成功建立TDI哮喘小鼠模型。

A modified method for establishing TDI-induced murine asthma model

Objective To establish the model of TDI asthma and to improve method of intra-tracheal instillation,and to evaluate the establishment of the model.Methods A total of 20 SPF grade healthy male BALB/c mice were randomly divided into TDI asthma group and solvent control group (acetone/olive oil),10 animals in each group.In the asthma group,the mice were dermally treated with 0.3％ TDI dropped on the dorsum of both ears (20 μ1 per ear) on days 1 and 8.On days 15,the mice were challenged with 20 μl 0.01％ TDI by the trachea.The solvent control group mice were sensitized and challenged by the same procedures with the same amount of solvent.After sensitization,the bronchoalveolar lavage fluid (BALF) from six mice in each group were collected.The inflammatory cells in BALF were counted and classified,IL-4 and IFN-γ levels in BALF supernatant were measured by ELISA,the pathological'changes of lung tissues were observed by HE stainingand response of mice were taken as the indexes to observe the viability of the replication.Results The asthmatic group demonstrated the symptoms of acute asthma,such as breathing deeply and fast,back bowing,forelimbs lifting,stool incontinence and so on.Compared with the control group (AOO group),in asthma group (TDI group) the total white blood cells,neutrophils,lymphocytes,monocytes,eosinophils,basophils in mice BALF and the levels of IL-4,IFN-γ in BALF supernatant were all significantly increased (P＜0.05).HE staining showed obvious inflammatory lesions in TDI asthmatic mice lung tissue.Conclusion Stimulating performed by tracheal instillation,the TDI asthma mouse model has been established in the present study.