TDI致哮喘小鼠肺组织中ROR-γt、IL-17A和Foxp3基因表达及其启动子区甲基化变化

发布时间：2018-04-27 13:46

本文选题：甲苯二异氰酸酯 + 哮喘　；参考：《中国职业医学》2017年01期

【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(P0.01),淋巴细胞百分比和单核细胞百分比均下降(P0.01)。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(P0.01),其启动子区Cp G位点3、4、5、6、8、11和12的甲基化程度均下降(P0.05);IL-17A mRNA相对表达水平升高(P0.01),其启动子区Cp G位点6、7的甲基化程度均下降(P0.01);Foxp3 mRNA相对表达水平下降(P0.01),其启动子区Cp G位点1和10的甲基化程度均升高(P0.01)。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能参与了Th17/Treg细胞免疫失衡。
[Abstract]:Objective to study the gene expression and methylation of retinoic acid-associated nuclear orphan receptor (ROR- 纬 T), interleukin (IL) -17A and fork head / winglike helix protein (3Foxp3) during asthma induced by toluene diisocyanate (TDI). Methods healthy male BALB/c mice without specific pathogens were randomly divided into asthma group and control group with 20 mice in each group. Mice in asthmatic group were sensitized with 20 渭 L TDI at the first and eighth days, respectively. The mice in the control group were sensitized and stimulated with 20 渭 L TDI at the mass ratio of 20 渭 L on the 15th day, and the mice in the control group were sensitized and stimulated with the same volume solvent on the 15th day. The histopathological changes of trachea and lung were observed 24 hours after stimulation. The inflammatory cell count and classification were collected from bronchoalveolar lavage fluid (BALF). The levels of IL-4 and IFN- 纬 in the supernatant of BALF were detected by enzyme-linked immunosorbent assay (Elisa). The mRNA expression of ROR- 纬 ttttthl IL-17A and Foxp3 in lung tissue was detected by real-time fluorescence quantitative polymerase chain reaction, and the methylation degree of ROR- 纬 ttttthl IL-17A and Foxp3 gene promoter region in lung tissue was detected by Mass Array flight mass spectrometry (FMS). Results the asthmatic symptoms such as wheezing, shortness of breath, nodding breathing, arched back and forelimb lifting were found in asthmatic mice after sensitization by TDI, but no such symptoms were found in the control group. Hematoxylin-eosin staining showed obvious inflammatory changes in trachea and lung of asthmatic mice. Compared with the control group, the total number of leukocytes, the percentage of neutrophils, the percentage of eosinophil and the level of IFN- 纬 in BALF of asthmatic mice were increased, while the percentage of lymphocytes and monocytes were decreased. Compared with the control group, The relative expression level of ROR- 纬 t mRNA in lung tissues of asthmatic mice was increased (P 0.01), and the methylation degree of CpG locus 3PG was decreased, and the relative expression of IL-17A mRNA was increased (P0.01A). The methylation degree of CP G locus 67 in the promoter region of asthma group was lower than that of CpG site 67. The methylation degree of CpG locus 67 in the promoter region of asthmatic mice was significantly lower than that of CpG site 67. The relative expression level of Foxp3 mRNA in P0.01 was decreased and the methylation degree of CpG loci 1 and 10 increased in the promoter region. Conclusion in the pathogenesis of asthma induced by TDI, the imbalance of Th17/Treg cellular immunity and the abnormal methylation of IL-17A and Foxp3 promoter region of ROR- 纬 ttttttttttttttttths may play an important role in the imbalance of Th17/Treg cell immunity.
【作者单位】：济南大学山东省医学科学院医学与生命科学学院;山东省医学科学院山东省职业卫生与职业病防治研究院;
【基金】：国家自然科学基金面上项目(81470145) 国家十二五科技支撑计划项目(2014BAI12B02) 中国博士后科学基金面上项目(2014M551951) 山东省科技发展计划(2012G0030034) 山东省医药科技卫生计划项目(2014WS0334,2014WS0332)
【分类号】：R562.25

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