CAJ | 학술논문

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.
목적 관찰이양화규(SiO2)대폐상피세포간질전화중E개점단백(E-cadherin)、α-평활기격동단백(α-SMA)화전화생장인자β1(TGF-β1)표체적영향,병탐토표피생장인자수체(EGFR)신호통로재폐섬유화상피간질전화중적작용.방법 용50 μg/ml적SiO2분진자격폐포거서세포(AM),분별작용3、6、12、18、24、36 h후수집상청액,용ELISA방법검측상청중TGF-β1단백적표체,병이TGF-β1표체적고봉시간점(T18h)적AM배양상청작위조건배양상청.용종농도분별위0(대조조)、50(50 μg/mlSiO2)、100(100 μg/ml SiO2)、200 μg/ml(200 μg/ml SiO2)적SiO2분진여AM조건배양상청액(T18 h)공동자격폐상피세포,부육48 h후,우도치현미경하관찰세포형태학개변,병수집불동시단세포,용반전록취합매련반응(RT-PCR)검측E-cadherin、α-SMA、EGFR mRNA표체수평,세포면역형광방법검측E-cadherin、α-SMA급EGFR단백표체적변화.결과 염진조AM배양상청액중TGF-β1단백적표체재각시간점균명현고우대조조,차수작용시간적연장정선승고후강저적추세,18h시수평최고,차이유통계학의의(P＜0.05).도치현미경관찰폐상피세포경SiO2처리후세포형태출현간질세포특점,유아란석상전변위방추형혹사형,위족양개변,사성섬유세포,SiO2 200 μg/ml조최명현.수착SiO2농도적승고,E-cadherin mRNA화단백표체축점하조,재200 μg/ml SiO2조표체최저,α-SMA、EGFR mRNA화단백표체축점상조,200 μg/ml SiO2조α-SMA표체최고;50、100、200 μg/ml SiO2조여대조조적차이유통계학의의(P＜0.05).결론 SiO2체외자격AM,사TGF-β1단백표체수평명현승고,기상청액여SiO2가공동유도폐상피세포향간질세포전화,기궤제가능여EGFR신호통로유관.
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) and the control group (P＜0.05).Conclusion After being stimulated by SiO2 in vitro,AMs have significantly increased expression level of TGF-β1 protein.The AM supernatant together with SiO2 can induce the transition of pulmonau epithelial cells to mesenchvmal cells,and its mechanism may be related to the EGFR signaling pathway.