二氧化硅致肺上皮细胞间质转化中表皮生长因子受体信号通路的作用  

Roles of epidermal growth factor receptor signaling pathway in silicon dioxide-induced epithelial-mesenchymal transition in human pulmonary epithelial cells

作　　者：宋文文[1] 张正福[2] 肖华 孙少杰 张华 

机构地区：[1] 即墨市人民医院呼吸科,266200 [2] 青岛市肿瘤医院放射科 [3] 青岛市中心医疗集团职业病防治院 [4] 青岛市中心医疗集团职业病防治院普外科

出　　处：《中华劳动卫生职业病杂志》2015年第9期663-667,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：2013年青岛市民生计划项目

摘　　要：目的 观察二氧化硅（SiO2）对肺上皮细胞间质转化中E钙黏蛋白（E-cadherin）、α-平滑肌激动蛋白（α-SMA）和转化生长因子β1（TGF-β1）表达的影响,并探讨表皮生长因子受体（EGFR）信号通路在肺纤维化上皮间质转化中的作用.方法 用50 μg/ml的SiO2粉尘刺激肺泡巨噬细胞（AM）,分别作用3、6、12、18、24、36 h后收集上清液,用ELISA方法检测上清中TGF-β1蛋白的表达,并以TGF-β1表达的高峰时间点（T18h）的AM培养上清作为条件培养上清.用终浓度分别为0（对照组）、50（50 μg/mlSiO2）、100（100 μg/ml SiO2）、200 μg/ml（200 μg/ml SiO2）的SiO2粉尘与AM条件培养上清液（T18 h）共同刺激肺上皮细胞,孵育48 h后,于倒置显微镜下观察细胞形态学改变,并收集不同时段细胞,用反转录聚合酶链反应（RT-PCR）检测E-cadherin、α-SMA、EGFR mRNA表达水平,细胞免疫荧光方法检测E-cadherin、α-SMA及EGFR蛋白表达的变化.结果 染尘组AM培养上清液中TGF-β1蛋白的表达在各时间点均明显高于对照组,且随作用时间的延长呈先升高后降低的趋势,18h时水平最高,差异有统计学意义（P＜0.05）.倒置显微镜观察肺上皮细胞经SiO2处理后细胞形态出现间质细胞特点,由鹅卵石状转变为纺锤形或梭型,伪足样改变,似成纤维细胞,SiO2 200 μg/ml组最明显.随着SiO2浓度的升高,E-cadherin mRNA和蛋白表达逐渐下调,在200 μg/ml SiO2组表达最低,α-SMA、EGFR mRNA和蛋白表达逐渐上调,200 μg/ml SiO2组α-SMA表达最高;50、100、200 μg/ml SiO2组与对照组的差异有统计学意义（P＜0.05）.结论 SiO2体外刺激AM,使TGF-β1蛋白表达水平明显升高,其上清液与SiO2可共同诱导肺上皮细胞向间质细胞转化,其机制可能与EGFR信号通路有关.Objective To investigate the effect of silicon dioxide （SiO2） on the expression of E-cadherin,α-smooth muscle actin （α-SMA）,and transforming growth factor β1 （TGF-β1） in human pulmonary epithelial cells （A549） with epithelial-mesenchymal transition （EMT）,and to study the roles of epidermal growth factor receptor （EGFR） signaling pathway in SiO2-induced EMT in A549 cells in vitro.Methods Alveolar macrophages （AMs） were stimulated with 50 μg/ml SiO2 for 3,6,12,18,24,or 36 h,and the supernatants were collected to measure the expression of TGF-β1 protein by ELISA.The AM supernatant in which TGF-β1 reached the highest expression （T=18 h） was used as AM-conditioned supernatant.A549 cells were cultured in AM-conditioned supernatant and stimulated with indicated doses of SiO2 （0, 50,100,and 200 μg/ml） for 48 h.The cell morphological changes were observed using an inverted microscope.The cells were collected at different times,and the mRNA and protein expression levels of E-cadherin,α-SMA,and EGFR were measured bv RT-PCR and immunocytofluorescence,respectively.Results After stimulation by SiO2,the expression level of TGF-β1 protein at each time point was significantly higher in the presence of AM supernatants than in the absence of AM supernatants （P〈0.05）.With the action time,the expression level of TGF-β1 protein increased at first and then decreased,and the highest level was reached at 18 h.After exposure to SiO2,A549 cells exhibited mesenchymal characteristics,such as a spindle shape,pseudopodia change,and fibroblast-like morphology,as observed by inverted microscope,especially in the 200 μg/ml group.With increased concentration of SiO2,the mRNA and protein expression of E-cadherin was down-regulated gradually,especially in the 200 μg/ml group,whereas the mRNA and protein expression of α-SMA and EGFR was up-regulated gradually,especially in the 200 μ g/ml group.There were significant differences between the SiO2-treated groups （50,100,and 200 μg/ml SiO2） an

关 键 词：二氧化硅 受体 表皮生长因子 肺泡 上皮细胞 

分 类 号：R135.2[医药卫生—劳动卫生]