中文摘要：

目的研究甲苯二异氰酸酯（TDI）致哮喘过程中维甲酸相关核孤儿受体-γt（ROR-γt）、白细胞介素（IL）-17A和叉头状/翅状螺旋蛋白3（Foxp3）基因表达及其启动子区甲基化变化情况。方法无特定病原体级健康雄性BALB/c小鼠随机分为哮喘组和对照组,每组20只。哮喘组小鼠于第1和8天于耳背分别涂抹20μL质量体积比为0.30%的TDI致敏,第15天经气管予20μL质量体积比为0.01%的TDI激发;对照组小鼠予等体积的溶剂进行致敏与激发。激发后24 h,观察小鼠气管和肺组织病理学变化情况,收集支气管肺泡灌洗液（BALF）进行炎性细胞计数与分类,以酶联免疫吸附实验法检测BALF上清液中IL-4和干扰素-γ（IFN-γ）的水平,以实时荧光定量聚合酶链式反应法检测肺组织中ROR-γt、IL-17A和Foxp3的mRNA表达,以Mass Array飞行质谱法检测肺组织中ROR-γt、IL-17A和Foxp3基因启动子区甲基化程度。结果经TDI致敏激发后,哮喘组小鼠均出现明显的喘息、呼吸急促、点头呼吸、弓背、前肢缩抬等哮喘症状,对照组小鼠无上述症状。苏木素-伊红染色可见哮喘组小鼠气管和肺组织均出现明显炎性病变。与对照组比较,哮喘组小鼠BALF中白细胞总数、中性粒细胞百分比、嗜酸性粒细胞百分比、IL-4水平和IFN-γ水平均升高（P〈0.01）,淋巴细胞百分比和单核细胞百分比均下降（P〈0.01）。与对照组比较,哮喘组小鼠肺组织中ROR-γt mRNA相对表达水平升高（P〈0.01）,其启动子区CpG位点3、4、5、6、8、11和12的甲基化程度均下降（P〈0.05）;IL-17A mRNA相对表达水平升高（P〈0.01）,其启动子区CpG位点6、7的甲基化程度均下降（P〈0.01）;Foxp3 mRNA相对表达水平下降（P〈0.01）,其启动子区CpG位点1和10的甲基化程度均升高（P〈0.01）。结论 TDI所致哮喘发病过程中存在Th17/Treg细胞免疫失衡;ROR-γt、IL-17A和Foxp3基因启动子区甲基化异常可?

英文摘要：

Objective To explore the changes of retinoic acid related orphan receptor-γt（ ROR-γt）,interleukin（ IL）-17 A and forkhead / winged helix protein 3（ Foxp3） mRNA expression and promoter methylation in the process of asthma induced by toluene-diisocyanate（ TDI）. Methods Specific pathogens free grade healthy male BALB / c mice were randomly assigned into asthma group and control group with 18 animals in each group. In the asthma group,the mice were sensitized with 0. 30% TDI（ mass-volume concentration） dropped on the dorsum of both ears（ 20 μL / ear） on day 1 and day 8. On day 15,the mice were challenged with 20 μL 0. 01% TDI（ mass-volume concentration） by the trachea. The control group mice were sensitized and challenged by the same procedures with the same amount of solvent（ acetone / olive oil）. The mice were challenged 24 hours,the pathological changes of trachea and lung tissues were observed. The bronchoalveolar lavage fluid（ BALF） from each group was collected,and the inflammatory cells in BALF were counted and classified. IL-4and Interferon-γ（ IFN-γ） levels in BALF supernatant were measured by enzyme-linked immunosorbent assay. ROR-γt,IL-17 A and Foxp3 mRNA expression in the lung tissue were measured by real-time fluorescent quantitative polymease chain reaction. The degree of ROR-γt,IL-17 A and Foxp3 promoter methylation in lung issue were detected by Mass Array system.Results The asthmatic group demonstrated the symptoms of acute asthma,such as breathing deeply and fastly,bowing the back,lifting the forelimbs,et al. But the control group had no such symptoms in mice. Hematoxylin-Eosin staining showed obvious inflammatory lesions in the trachea and lung tissue of asthmatic mice. Compared with the control group,the white blood cell count,the neutrophil and eosinophil percentages in BALF,the IL-4,IFN-γ levels in BALF supernatant in asthma group were all significantly increased（ P〈0. 01）,meanwhile the lymphocyte and monocyte percentages in BALF were reduced?