The Regulating Effect Of Substate Stiffess On Cytoskeletal Protein And Cellular Elasticity Modulus

As a complicated macromolecular network,extracellular matrix(ECM)is featured with the unique physical property,biochemical characteristics and mechanical property,which can affect cellular biological behaviors directly and indirectly and become essential factors.When metabolism of ECM takes place anomaly,organizational structure and mechanical property of ECM are changed,accompanying with some diseases,such as tissue fibrosis,proliferation and migration of cancer cells,and epithelial-mesenchymal transition,thus it shows that microenvironment stiffness of cells plays an important role on cancer generation,migration and deterioration.To study how to perceive surrounding tissue stiffness and response disciplines of hardness in cancer cells can provide valuable reference for generation mechanism of cancer,assist and improve the therapeutic schedule.This paper mainly studied influences of substrate with different stiffness on biomechanical characteristics of cells.The water-soluble polymer with good biological compatibility—polyacrylamide hydrophilicgel was applied as the cultivation substrate to simulate extracellular environment.By adjusting the proportion between acrylamide and Methylene bisacrylamide,elastic substrate with different stiffness was prepared.Cervical cancer cells in logarithmic phase were inoculated into1 KPa,10KPa and 150 KPa of polyacrylamide hydrophilic gel substrate to cultivate for 24-48 h.The cultivated cervical cancer cells were conducted the following experiment to observe influences of substrate stiffness on behavioral characteristics of cellular biomechanics:(1)Molecular motor protein in cytoskeleton was measured,including myosin ? distribution and motion rate.Cervical cancer cells could stably transfect fluorescent myosin ?.Laser scanning confocal microscope was used to measure fluorescent distribution of cervical cancer cells' fluorescent myosin ? along cellular long axis growing on three kinds of stiffness substrates.Fluorescence recovery after photobleaching was utilized to measure recovery rate of fluorescent myosin ? on the cellular terminal.(2)Distribution of actin filament in the entire cell was measured.Phalloidin-Tetramethylrhodamine B Isothiocyanate was applied to do fluorescence staining on cervical cancer cells' actin filament and measure actin filament distribution of cervical cancer cell's actin filament cultivated on three kinds of stiffness.(3)Micropipette aspiration technique was applied.By combiningwith semi-infinite body model,cellular elasticity modulus of the single cervical cancer cells under the spreading state was measured.Results showed that cervical cancer cells grown in substrate with different stiffness were presented in distribution differences of myosin ?and actin filament.As for cervical cancer cells grown on the soft substrate,there was a little myosin ? and actin filament on the cell edge.Motion rate of myosin ? was slow and cellular elasticity modulus was reduced.As for cervical cancer cells grown on the hard substrate,myosin ? and actin filament were presented in the obvious coherent condition of cell edge.Cellular elasticity modulus was also obviously higher than cells grown on soft and hard substrates.Experimental results showed that substrate hardness would obviously change influences of important cytoskeletal protein effects on cellular elasticity modulus,such as myosin? and actin filament and it could affect cellular state of cancer cells under different microenvironment.