發(fā)布時(shí)間：2018-04-27 13:46

  本文選題：甲苯二異氰酸酯 + 哮喘��； 參考：《中國(guó)職業(yè)醫(yī)學(xué)》2017年01期

【摘要】：目的研究甲苯二異氰酸酯(TDI)致哮喘過(guò)程中維甲酸相關(guān)核孤兒受體-γt(ROR-γt)、白細(xì)胞介素(IL)-17A和叉頭狀/翅狀螺旋蛋白3(Foxp3)基因表達(dá)及其啟動(dòng)子區(qū)甲基化變化情況。方法無(wú)特定病原體級(jí)健康雄性BALB/c小鼠隨機(jī)分為哮喘組和對(duì)照組,每組20只。哮喘組小鼠于第1和8天于耳背分別涂抹20μL質(zhì)量體積比為0.30%的TDI致敏,第15天經(jīng)氣管予20μL質(zhì)量體積比為0.01%的TDI激發(fā);對(duì)照組小鼠予等體積的溶劑進(jìn)行致敏與激發(fā)。激發(fā)后24 h,觀察小鼠氣管和肺組織病理學(xué)變化情況,收集支氣管肺泡灌洗液(BALF)進(jìn)行炎性細(xì)胞計(jì)數(shù)與分類(lèi),以酶聯(lián)免疫吸附實(shí)驗(yàn)法檢測(cè)BALF上清液中IL-4和干擾素-γ(IFN-γ)的水平,以實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)法檢測(cè)肺組織中ROR-γt、IL-17A和Foxp3的mRNA表達(dá),以Mass Array飛行質(zhì)譜法檢測(cè)肺組織中ROR-γt、IL-17A和Foxp3基因啟動(dòng)子區(qū)甲基化程度。結(jié)果經(jīng)TDI致敏激發(fā)后,哮喘組小鼠均出現(xiàn)明顯的喘息、呼吸急促、點(diǎn)頭呼吸、弓背、前肢縮抬等哮喘癥狀,對(duì)照組小鼠無(wú)上述癥狀。蘇木素-伊紅染色可見(jiàn)哮喘組小鼠氣管和肺組織均出現(xiàn)明顯炎性病變。與對(duì)照組比較,哮喘組小鼠BALF中白細(xì)胞總數(shù)、中性粒細(xì)胞百分比、嗜酸性粒細(xì)胞百分比、IL-4水平和IFN-γ水平均升高(P0.01),淋巴細(xì)胞百分比和單核細(xì)胞百分比均下降(P0.01)。與對(duì)照組比較,哮喘組小鼠肺組織中ROR-γt mRNA相對(duì)表達(dá)水平升高(P0.01),其啟動(dòng)子區(qū)Cp G位點(diǎn)3、4、5、6、8、11和12的甲基化程度均下降(P0.05);IL-17A mRNA相對(duì)表達(dá)水平升高(P0.01),其啟動(dòng)子區(qū)Cp G位點(diǎn)6、7的甲基化程度均下降(P0.01);Foxp3 mRNA相對(duì)表達(dá)水平下降(P0.01),其啟動(dòng)子區(qū)Cp G位點(diǎn)1和10的甲基化程度均升高(P0.01)。結(jié)論 TDI所致哮喘發(fā)病過(guò)程中存在Th17/Treg細(xì)胞免疫失衡;ROR-γt、IL-17A和Foxp3基因啟動(dòng)子區(qū)甲基化異�？赡軈⑴c了Th17/Treg細(xì)胞免疫失衡。
[Abstract]:Objective to study the gene expression and methylation of retinoic acid-associated nuclear orphan receptor (ROR- 緯 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- 緯 in the supernatant of BALF were detected by enzyme-linked immunosorbent assay (Elisa). The mRNA expression of ROR- 緯 ttttthl IL-17A and Foxp3 in lung tissue was detected by real-time fluorescence quantitative polymerase chain reaction, and the methylation degree of ROR- 緯 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- 緯 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- 緯 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- 緯 ttttttttttttttttths may play an important role in the imbalance of Th17/Treg cell immunity.
【作者單位】： 濟(jì)南大學(xué)山東省醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與生命科學(xué)學(xué)院;山東省醫(yī)學(xué)科學(xué)院山東省職業(yè)衛(wèi)生與職業(yè)病防治研究院;
【基金】：國(guó)家自然科學(xué)基金面上項(xiàng)目(81470145) 國(guó)家十二五科技支撐計(jì)劃項(xiàng)目(2014BAI12B02) 中國(guó)博士后科學(xué)基金面上項(xiàng)目(2014M551951) 山東省科技發(fā)展計(jì)劃(2012G0030034) 山東省醫(yī)藥科技衛(wèi)生計(jì)劃項(xiàng)目(2014WS0334,2014WS0332)
【分類(lèi)號(hào)】：R562.25

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