The Co-stimulation Effects Of Fluid Shear Stress And Material Surface Chemical On The Behavior Of Human Umbilical Vein Endothelial Cells

Objective:Human umbilical vein endothelial cells（HUVECs）were stimulated by fluid shear stress and chemical functional groups on the surface of materials.The response of HUVECs to gradient fluid shear stress was observed,and the effect of material chemistry on the response of HUVECs to shear stress was explored.Methods:The self-assembled monolayers with-OH、-CH₃ and-NH₂ as terminal chemical functional groups were prepared on the surface of Glass slides using the self-assembled monolayer technology.Using blank Glass slides were used as the control,and the materials were characterized by detecting the water contact Angle on the surface of the materials.HUVECs were seeded on Glass,-OH,-NH₂ and-CH₃ slides respectively.ATP release was detected within 15minutes and no release was detected within 1 hour.The expression of endothelial nitric oxide synthase（e NOS）was detected by Western blotting after 1 hour of culture.Formation of adhesion spots（FAS）and cytoskeleton were observed by confocal laser scanning microscope after 24 hours,and the total protein content was determined by BCA method.Build a shear force stimulation platform based on the existing parallel plate flow chamber.HUVECs were respectively seeded on the surface of 4 groups of glass slides,when the cells fused to 80%,they were loaded with 5 dyn/cm²,15 dyn/cm²and 20 dyn/cm² fluid shear stress（FSS）respectively for 1 hour.ATP release was detected within 15 minutes and NO release was detected within 1 hour.The expression of e NOS was detected by Western blotting and the total protein content was determined by BCA.Results:Through the detection of water contact Angle,it is proved that the self-assembled monolayer technology has successfully grafted onto the surface of the glass with strong hydrophilic-OH chemical groups,moderate hydrophilic/hydrophobic-NH₂ chemical groups and strong hydrophobic-CH₃chemical groups.HUVECs were seeded on materials with different chemical functional groups on the surface.There was no significant difference in the responses of HUVECs in each group under the chemical stimulation of the material surface alone.Compared with the cytoskeleton and adhesion plaque of endothelial cells on Glass surface,the hydrophilicity and hydrophobicity of-NH₂were moderate,and the endothelial cells formed the best adhesion quality cytoskeleton and adhesion plaque on-NH₂ glass slide;the strong hydrophilic-OH and strong hydrophobic-CH₃ groups were not conducive to cell adhesion,while the poor adhesion quality cytoskeleton and adhesion plaque were formed on-OH and-CH₃ glass slide.On the basis of the existing parallel plate flow chamber device,a shear force stimulation platform was successfully built.After loading gradient shear force on the surface of the material to chemically co-stimulate endothelial cells,it was found that low shear force（5dyn/cm²）did not cause endothelial cells on Glass and-NH₂ to respond,but low shear force caused a strong response of-OH and-CH₃;Moderate shear force（15dyn/cm²）and high shear force（20dyn/cm²）caused the response of all groups of cells.Among them,the response level of-NH₂ group was the strongest,with the highest level,followed by the Glass group.The response level of-OH and-CH₃ group was equal and in the lowest state.In the-OH and-CH₃ groups,when the shear force gradient is stimulated,the response is the strongest at low shear force,followed by medium shear force,and the weakest is high shear force.Conclusion:The chemical functional groups on the surface of the material affect the quality of endothelial cell adhesion and cytoskeleton,and change the initial threshold of HUVECs to respond to FSS stimulation and the shear force threshold for cells to produce the best response,so that the response state of HUVECs to shear stress stimulation was changed.Focal adhesion and cytoskeleton may be the interaction sites of physical shear stress and material chemical stimulation.