| The changes of mechanical microenvironment have played an important role in the development of many diseases.In response to changes in the mechanical microenvironment,cells can activate multiple intracellular signaling pathways.The activation of these signaling pathways plays an important role in determining cell fate.Pulmonary fibrosis disease is a disease with high morbidity and mortality.Its main features are the substantial destruction of lung tissue and abnormal excessive deposition of extracellular matrix,which leads to changes in the mechanical microenvironment of extracellular matrix.At present,the main cause of pulmonary fibrosis is still unknown and lacks effective treatments.It has been found that epithelial-mesenchymal transition(EMT)plays an important role in the development of lung fibrosis diseases,and a large number of lung fibroblasts can be transformed by epithelial-mesenchymal processes from epithelial cells.However,how the changes in the mechanical microenvironment affect epithelial-mesenchymal transition and thus regulate the development of pulmonary fibrotic diseases are still unclear.Based on this problem,this study used human alveolar epithelial cells A549 as experimental subject,and used PDMS micropillar arrays substrate as the base materials.This study aimed to discuss the effects of changes in the topology of mechanical microenvironment on the biological behavior and EMT process of alveolar epithelial cells,and to study the regulation of Smad-related signaling pathways and FAK signaling pathways in this process.Firstly,a microfabricated PDMS micropillar array structure substrate with a nominal diameter of 10 ?m,a nominal pitch of 2 ?m,4 ?m and 7 ?m,and a nominal height of 4 ?m was fabricated.Secondly,the modification and mechanical detection of the PDMS micropillar array structural substrate were performed and its composite culture with A549 cells was realized.Again,A549 cells were seeded on different planes and different micropillar array structures to examine the effect of micropillar array structure on the biological behavior of A549 cells such as morphology,spreading area,adhesion protein expression,and framework rearrangement.Then,TGF-?1 at a concentration of 10 ng/mL induces epithelial-mesenchymal transition in A549 cells,and the effect of micropillar array-based substrate on TGF-?1-induced epithelial-mesenchymal transition is examined.Finally,the possible mechanism of micropillar array structure substrate on the transformation process of epithelial mesenchymal was preliminarily explored.We detected the expression of the corresponding protein in the Smad-related signaling pathway,and then used the method of stable gene silencing by the lentivirus to stably silence the A549.The focal adhesion kinase(FAK)gene in the cell,and the effect of silencing of this gene on the epithelial mesenchymal transition process affected by the micropillar array structure of the substrate.The main experimental content and results of this article are as follows:(1)There was no significant difference in the Young's moduli between the PDMS planar substrate and the micropillar array substrate under the same hardness conditions.Both the PDMS planar substrate and the micropillar array-type substrate could be complexed with A549 cells after being lining with fibronectin.(2)The scanning electron microscopy was used to study the effect of micropillar array structure on the morphology of A549 cells,which shows that micropillar array structure had a very significant effect on the morphology of A549 cells when cells grow on dense micropillars.The cells tended to float on the top of the micropillar,and the cell roundness and spreading area were large.With the increase of the spacing of the micropillars,the spreading area and roundness of the cells reduced,and the polarization behavior of cells became apparent.It is more obvious that epithelial cells began to have the morphology of mesenchymal cells.Immunofluorescence technique and Western Blot were used to examine the effect of the micropillar array structure on the cytoskeleton and focal adhesion protein.It was found that the presence of the micropillar disrupted the order of the cytoskeleton distribution compared with the cells on a planar substrate.The arrangement of the skeleton filaments was neat,and as the spacing of the micropillars increases,the expression of the adhesion protein began to increase,and the cell adhesion began to increase.(3)Western Blot technique was used to study the effect of micropillar array structure on the TGF-?1-induced EMT process and the expression of potentially relevant signaling pathway proteins.It was found that the micropillar array topology could affect the EMT process with micro with the increase of the pillar spacing,the expression of Vimentin protein increased significantly.Results suggested that the topology could synergize with TGF-?1 growth factor to promote the transformation of A549 cells into mesenchymal cells.The process was related to the Smad signaling pathway.(4)The lentivirus was used to stably silence the FAK gene and examine the effect of the gene on the EMT process that affects the micropillar array structure.The study found that the FAK gene was closely related to the EMT process influenced by the micropillar array structure.The silencing of the FAK gene weakened the influence of the micropillar array structure on the EMT process,which suggests that the FAK-related signaling pathway affected the EMT of the mechanical microenvironment.The process played an important role.This experimental study confirmed that the micropillar array structure substrate could affect the biological behavior of A549 cells and participate in the regulation of EMT process.The results of the study will help us to deepen our understanding of the mechanical microenvironment to regulate the process of pulmonary fibrosis and provide research foundation and data support for the treatment of pulmonary fibrosis disease.It can also provide a target for the prevention and treatment of pulmonary fibrosis. |
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