【摘要】 目的：探讨灭活母牛分枝杆菌雾化吸入对哮喘小鼠气道杯状细胞化生和黏液高分泌的影响。方法：将24只雄性Balb/c小鼠随机分为对照组、哮喘组、药物干预组和DAPT干预组，每组6只。哮喘组、药物干预组、DAPT干预组小鼠用鸡卵清蛋白（OVA）建立小鼠哮喘模型，药物干预组雾化吸入2.25μg/mL灭活母牛分枝杆菌，DAPT干预组小鼠腹腔注射γ-分泌酶抑制剂DAPT(2S)-N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰]-2-苯基甘氨酸叔丁酯（0.5 mg/kg）阻断Notch通路信号，第21、第22、第23、第24、第25天，哮喘组、药物干预组、DAPT干预组小鼠给予OVA溶液（1 mg OVA/mL PBS）雾化吸入30 min，正常组雾化等量PBS 30 min；行无创气道反应性检测，病理HE、PAS染色观察小鼠肺组织病理改变和气道黏液分泌情况，免疫组织化学染色观察小鼠肺组织杯状细胞分泌蛋白Muc5ac、clara细胞分泌蛋白CCSP的表达；实时荧光定量聚合酶链式反应（RT-qPCR）检测clara细胞标志物Scgb1a1和Muc5ac mRNA表达；蛋白免疫印迹法（Western blotting）检测Notch3、Hes1、Muc5ac、CCSP蛋白表达。结果：与对照组相比，哮喘组小鼠气道反应性升高，肺组织炎症细胞浸润，杯状细胞化生和黏液分泌明显增多，肺组织Muc5ac m RNA、Notch3、Hes1、Muc5ac蛋白表达升高，Scgb1a1 mRNA和CCSP蛋白表达降低（P<0.05）。与哮喘组相比，药物干预组和DAPT干预组小鼠气道反应性降低，肺组织炎症细胞浸润、杯状细胞化生和黏液分泌减少，肺组织Muc5ac mRNA、Notch3、Hes1、Muc5ac蛋白表达降低，Scgb1a1 m RNA和CCSP蛋白表达增高（P<0.05）。结论：灭活母牛分枝杆菌雾化吸入对哮喘小鼠气道高反应性、炎症和黏液高分泌有改善作用，其机制可能与调控Notch3/hes1通路抑制clara细胞向杯状细胞分化有关。更多还原

【Abstract】 Objective:To investigate the effects of aerosol inhalation of inactivated Mycobacterium vaccae on airway goblet cell metaplasia and mucus hypersecretion in asthmatic mice.Methods:Twenty-four Balb/c male mice were randomly divided into 4 groups:control group,asthma group,drug intervention group and DAPT intervention group,with 6 mice in each group.Mice in asthma group,drug intervention group and DAPT intervention group were treated with ovalbumin (OVA) to establish asthma model.The drug intervention group mice were inhaled with 2.25μg/mL inactivated Mycobacterium vaccae.The DAPT intervention group mice were intraperitoneally injected with 0.5mg/kg γ-secretase inhibitor(DAPT (2S)-N-[N-(3,5-difluorophenyl)-L-alanyl]-2-phenylglycine tert-butyl ester) to block the Notch signaling pathway.On the 21st,22nd,23rd,24th and 25th days,mice in asthma group,drug intervention group and DAPT intervention group were given OVA solution (1 mg OVA/mL PBS) for 30 min atomization inhalation and the normal group was given the same amount of PBS for 30min.A noninvasive airway reactivity test was performed to assess airway responsiveness.HE staining and PAS staining of lung tissues were performed to observe the pathological changes and airway mucus secretion.The protein expressions of Muc5ac,CCSP were observed by immunohistochemistry staining.The mRNA expressions of Scgb1a1,Muc5ac were detected by RT-qPCR.The protein expressions of Notch3,Hes1,Muc5ac,CCSP were detected by Western blotting.Results:Compared with the control group,the airway reactivity,inflammatory cell infiltration,goblet cell proliferation,mucus secretion and the expressions of Muc5ac mRNA,Notch3,Hes1 and Muc5ac protein in asthma group significantly increased,while the protein expressions of CCSP and Scgb1a1 mRNA decreased (P<0.05).Compared with asthma group,the airway reactivity of mice,inflammatory cell infiltration,goblet cell proliferation,mucus secretion,the expressions of Muc5ac m RNA,Notch3,Hes1 and Muc5ac protein in lung tissues decreased in the drug intervention group and the DAPT intervention group,while the expressions of Scgb1a1 mRNA and CCSP protein increased (P<0.05).Conclusion:Aerosol inhalation of inactivated Mycobacterium vaccae can reduce airway hyperresponsiveness,inflammation and mucus hypersecretion in asthmatic mice,which may be related to the regulation of Notch3/HES1 pathway inhibiting clara cell differentiation into goblet cells.更多还原