γ干扰素诱导气道黏液细胞凋亡及其机制的实验研究

A study of interferon-γ induced airway mucous cell apoptosis and its mechanisms

目的研究γ干扰素(IFN-γ)对气道黏液细胞的作用及其机制.方法 (1)体外培养正常人气道上皮细胞5～7 d后,以含50 ng/ml的IFN-γ的培养基再培养3 d,采用异硫氰酸荧光素(FITC)免疫荧光和末端脱氧核苷酸转移酶介导的脱氧三磷酸尿嘧啶缺口末端标记(TUNEL)方法,观察IFN-γ诱导体外培养人气道黏液细胞凋亡及其机制.(2)20只野生型C57BL/6J小鼠经致敏后按数字表法随机分为4组,每组5只.分别经鼻气道内滴注IFN-γ 50 ng、IFN-γ 100 ng、白细胞介素13(IL-13)5 μg,并以生理盐水作对照,采用细胞形态学方法,分别计数各组致敏小鼠单位气道长度内黏液细胞数;采用TUNEL法检测气道黏液细胞的凋亡改变.结果 (1)免疫荧光细胞核染色联合TUNEL检测显示,经IFN-γ作用后的人气道黏液细胞出现凋亡现象,细胞形态改变,胞核破裂;在细胞凋亡过程中,凋亡诱导因子Bax表达增加,并向线粒体转位.(2)经鼻向气管内滴注100 ng IFN-γ后,小鼠气道单位长度(黏液细胞数/毫米气道基底层,下同)黏液细胞数量为(28±6)个/mm,生理盐水组为(58±12)个/mm,IL-13组为(59±6)个/mm,生理盐水组和IL-13组与IFN-γ 100 ng组比较差异有统计学意义(P<0.05);IFN-γ 50 ng滴注组[(48±11)个/mm]与生理盐水组[(58±12)个/mm]和IL-13组[(59±6)个/mm]比较,差异均无统计学意义(P均>0.05).TUNEL检测显示IFN-γ诱导致敏小鼠在体的气道黏液细胞凋亡.结论 IFN-γ诱导气道黏液细胞凋亡,该过程是通过Bax表达上调和Bax线粒体转位而实现.IFN-γ诱导气道黏液细胞凋亡在支气管哮喘治疗中具有意义.

Objective To investigate the effect of interferon-γ(IFN-γ) on airway mucous cells and its mechanisms. Methods (1)Normal human bronchial epithelial cells(NHBEs) were cultured under specific conditions,and treated by IFN-γ for 3 days. The cells were analyzed with fluorescein isothiocyanate(FITC) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay. (2)Twenty wild type C57BL/6J mice were immunized and divided into 4 groups,and treated with IFN-γ(50 ng and 100 ng,respectively),interleukin-13(IL-13,5 μg) and saline by nostril instillation. The mice were sacrificed and the airway mucous cells were analyzed by morphometry and TUNEL assay. Results (1)IFN-γ induced apoptosis in NHBEs,which showed condensed nuclei,nuclear and DNA fragmentaion,and were positive by TUNEL assay. Bax was upregulated and translocated from cell plasma to mitochondria under the treatment. (2)Airway mucous cell account in 100 ng IFN-γ instillation immunized mice group(28±6 mucous cells/mm basal lamina) was significantly decreased as compared to that in saline(58±12) and IL-13(59±6) instillation groups(all P<0.05). There was no difference among the IFN-γ 50 ng(48±11),saline(58±12) and IL-13(59±6) instillation groups(all P>0.05). TUNEL assay was also positive in airway mucous cells from IFN-γ instillation mice as compared to saline instillation mice. Conclusions IFN-γ leads to airway mucous cell apoptosis by Bax upregulating and translocation into mitochondria. This might be of significance in the new therapies of asthma.