RESUMEN
Objective: Construct 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. Methods: A549 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. Results: 1. 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. Conclusion: PQ 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.
