摘要
目的构建体外百草枯（paraquat，PQ）细胞纤维化模型，观察PQ对A549细胞中解整合素-金属蛋白酶17（ADAM17）表达的影响，探讨ADAM17在PQ中毒致肺纤维化中的作用。方法体外培养A549细胞，分为正常对照组、不同浓度PQ组，应用CCK-8检测细胞活力，筛选PQ浓度和时间，显微镜下观察细胞形态，ELISA测定各组纤维化标志物I型胶原（type I collagen，Col I）和纤连蛋白（fibronectin，FN）的表达，建立细胞纤维化模型；免疫细胞化学检测A549细胞中ADAM17的分布情况，RT-PCR和Western blot分别半定量检测ADAM17 mRNA及蛋白水平的表达情况。结果（1）随着PQ浓度增加及作用时间延长，A549细胞活力呈下降趋势，差异有统计学意义（P〈0.05），呈剂量依赖性和时间依赖性。（2）正常的A549细胞融合呈铺路石样生长，排列比较紧密，经PQ诱导后细胞排列较松散，细胞间连接变疏松，部分细胞溶解、死亡。（3）ELISA显示，随PQ浓度增加，Col I和FN表达增强，差异有统计学意义（P〈0.05）；随PQ时间延长，Col I和FN表达也逐渐增强，差异有统计学意义（P〈0.05），成功建立PQ细胞纤维化模型。（4）免疫细胞化学显示，ADAM17在A549细胞胞浆表达。（5）RT-PCR和Western blot表明，随着PQ浓度增加，ADAM17 mRNA及蛋白水平表达明显增加，差异有统计学意义（P〈0.05），以PQ 200 umol/L时最为明显。随着PQ作用时间延长，ADAM17 mRNA及蛋白表达水平也明显增加，差异有统计学意义（P〈0.05），在24 h达到高峰。结论百草枯可引起肺泡上皮细胞形态学改变，导致细胞损伤，成功建立细胞的纤维化模型，对A549细胞的毒性作用具有剂量和时间依赖性。ADAM17在PQ诱导的A549细胞中过表达，可能参与了百草枯诱导的肺纤维化过程。
ObjectiveConstruct a paraquat （PQ） cell fibrosis model in vitro, observe the effect of PQ on the expression of a disintegrin and metalloproteinase-17 （ADAM17） in A549 cells, and explore the role of ADAM17 in the pulmonary fibrosis induced by PQ poisoning.MethodsA549 cells are divided into normal control group, different concentration of PQ groups, CCK-8 is used to detect cell viability, screening concentration and time of PQ, cell morphology is observed under microscope; Enzyme-linked immunosorbent assay （ELISA） detectes fibrosis markers of collagen type I （Col I） and fibronectin （FN） expression. Establishment of cell model of fibrosis; distribution by immunocytochemical detection of ADAM17 in A549 cells, Reverse transcription-polymerase chain reaction and Western blot are used to detect the expression of ADAM17 mRNA and protein.Results1. With the increase of PQ concentration and the prolongation of the action time, the activity of A549 cells decreased （P〈 0.05） , which is dose-dependent and time dependent. 2.The normal A549 cells fusion is paving stone growth and arranged more closely. After PQ induction, the cell arrangement was loose, the intercellular connection became loose, and some cells dissolved and died. 3.ELISA showed that with the increase of PQ concentration, the expression of Col I and FN increased （P〈0.05） , and Col I and FN expression gradually increased with the prolongation of PQ time （P〈0.05） , and the fibroblast model is successfully established. 4. Immunocytochemistry showes that ADAM17 is expressed in the cytoplasm of A549 cells. 5. RT-PCR and Western blot showed that the expression of ADAM17 mRNA and protein increased significantly with the increase of PQ concentration （P〈0.05） , which is most obvious at PQ 200 μmol/L. With the prolonged action of PQ, the expression level of ADAM17 mRNA and protein also increased significantly （P〈0.05） , and reached the peak in 24 h.ConclusionPQ can induce morphological changes of alveolar epithelial cells, cause cell damage, and successfully establish a cell fibrosis model, which has a dose and time dependence on the toxicity of A549 cells. ADAM17 is overexpressed in the A549 cells induced by PQ and may be involved in the process of pulmonary fibrosis induced by paraquat.
作者
方文燕
林昌伟
王宝福
冯胜刚
Fang Wenyan;Lin Changwei;Wang baofu;Feng Shenggang(Graduate School of Luzhou Southwest Medical University, Luzhou 646000, Chin)
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2018年第1期12-17,共6页
Chinese Journal of Industrial Hygiene and Occupational Diseases
