| The spreading and migration of cells in the physiological and pathological processes such as injury repair and cancer metastasis all start from the local cytoskeleton protein,mechanical and morphological changes,and were always closely related.The cell-edge had active and significant deformation,which was one of the most direct manifestations of cell perception and response to the changes of microenvironment in vivo and in vitro.This process involved the regulation of intracellular cytoskeleton proteins and their regulatory proteins,cell mechanical properties,cell structure and morphology,and extracellular matrix properties.In the process of growth,migration and differentiation of cells in vivo,they all need to adjust their edges to adapt to the microenvironment and physiological characteristics in vivo.The study on the regulation mode and characteristics of intracellular and extracellular matrix in cell-edge regulation provided necessary support for dealing with the interaction between cell and microenvironment in tissue engineering and injury repair.In this paper,we focused on the biomechanics of the regulatory system of intracellular cytoskeleton protein and the microstructure of extracellular matrix.The actin-myosin system,include F-actin,myosin II,regulatory proteins Anillin and Rho A were analyzed.PDMS substrate was used to design micro channel structure to analyze the influence of micro channel structure on cell-edge.In terms of deformation,the data of cell area change and cell contour deformation were statistically analyzed.In terms of mechanical analysis,the surface elastic modulus of the cell-edge region was measured by atomic force microscopy,and the correlation with the above results was compared to analyze the biomechanical characteristics of cell-edge regulation.The main results and conclusions of this paper were as follows:(1)Mechanical changes caused by cytoskeleton proteins were most significant in cell-edge.Atomic force microscopy was used to focused on 14 areas along cell-edge over time,The ANLN gene was knocked down by Cytochalasin D,the actin fiber polymerization inhibitor,blebbsitatin,the ATPase activity inhibitor of myosin II and Calysulin A,respectively.It was concluded that the most significant region of cytoskeletal protein regulating cell-edge was the terminal edge of the long and short axis of cells.The results identified a key region for the analysis of cell-edge regulation,which laid a foundation for further work.(2)Regulation of cell-edge by cytoskeleton proteins.Firstly,the formation and aggregation of actin fibers and the contraction of myosin II were the main factors that enhance the surface elastic modulus of cell surface.Rho A activation enhanced the contraction and the cytoskeleton reorganization induced by ANLN knockdown also increase the surface elastic modulus.Secondly,we compared the mechanical properties of cells under changes of mechanical properties.It was believed that cells will balance the modulus trend change caused by actin and myosin through myosin II contractile activity provided by Rho A.When the modulus decreased,Rho A activates in cell-edge when the synthesis of actin fibers was inhibited.(3)Regulating effects of micro channel on cell-edge.Micro channel showed different regulatory effects on cell spreading and migration.First of all,the effect of micro channel on the change of cell area was mainly reflected in the phase of cell migration and deformation,and the cell area was significantly increased.Secondly,the morphology of the terminal edge of the long axis of the migrated cells was significantly changed by the micro channel,which was different from the classical planar cell migration.Specifically,the leading edge of the cells in the micro channel was elongated and protruded,and the trailing edge was regular ellipse.Finally,the differences of the moduli of the long axis of the cells were mainly reflected in the stable adhesion stage,and the differences of the polarity distribution of the moduli of the cells in the micro channel were greater.The results of this study had clarified the effects and stages of cytoskeleton and extracellular matrix micro-channel structure to cell-edge regulation,and provided a relatively clear standard for biomechanical analysis in subsequent studies.It provided a valuable experimental basis for the research and clinical treatment in the fields of wound repair,cancer metastasis and biomaterials. |
