Study On Mechanical Properties Of Endothelial Cells Based On Advanced Piezoelectric Technique And Its Combination With Fluorescence Microscopy

Vascular endothelial dysfunction is a key initiating factor in the development of atherosclerosis.Vascular endothelial permeability is a measurement index of endothelial barrier function.Factors inducing increase in endothelial cell permeability include cytoskeletal change,cell contraction,and cell-cell coupling.The damage of vascular permeability is just because of the broken balance between cell-cell and cell-extracellular matrix due to contractile force produced by the cytoskeleton,and is a major cause of inflammation.The gold standards currently used for endothelial cell permeability assessments are macromolecular permeability and transendothelial electrical resistance measurements.But it is more desirable to establish a method based on the direct measurements of the mechanical properties of endothelial cytoskeleton and cell contractility.At present,there is no suitable technique to measure the mechanical properties of vascular endothelial cells,including viscoelasticity and contractile or traction force,in a dynamic and continuous manner,which seriously affects the study and applications of cell mechanics in the detection of vascular barrier function and evaluation of drug effects.In this thesis,the surface stress or traction force during the adhesion of human umbilical vein endothelial cells(HUVECs)was quantitatively measured by the double resonator(AT and BT cut)quartz microbalance technique,and cell viscoelasticity index(CVI)was used to characterize the softness or stiffness of the cells;and drugs affecting the vascular barrier function were tested on their effects on the traction force and viscoelasticity of HUVECs.The results are summarized as follows:1)Cell traction force of HUVECs with different cell numbers(20,000-40,000)were quantitatively measured by 9 MHz AT and BT naked gold electrode deposited quartz crystals,indicating that the 30,000 cells generated the highest traction force without obvious cell-cell interactions.The gold electrodes of AT and BT cut quartz crystals were then modified with fibronectin to promote the adhesions of 30,000 HUVECs and used for testing the effects of two drugs affecting the endothelial cell barrier function,an agonist thrombin,and an antagonisty-27632 on the traction force and viscoelasticity of HUVECs.The results showed that the cells’traction force was increased and the cells became stiffer under the treatment of thrombin;whereas the cells’traction force decreased and cells became softer under the treatment of y-27632.2)Quartz crystal microbalance(QCM)combined with live cell imaging was used for the dynamic adhesions of different concentrations(1×10~4-4×10~4 cells)of HUVECs,the results showed that QCM frequency shift(Δf)and dynamic resistance change(ΔR)increased with the increase of HUVECs concentration,and the cell viscoelasticity index(CVI=ΔR/Δf)also increased with the increase of the cell concentration;indicating that the cells’cytoskeletal structure became more organized and stiffer at higher cell concentration,which was consistent with the results of fluorescent labeled stress fibers.The morphological changes of HUVECs cells were not significant before and after the treatments of thrombin and y-27632,but the changes of CVI were significant(increased and decreased respectively),suggesting that the cells became stiffer and softer;which indicates that the QCM signal is more sensitive than the optical signal.In order to achieve the simultaneous QCM determination and fluorescence microscopy observation for the cytoskeletons,the actin microfilaments of HUVECs cells,NRK rat kidney cells and H9C2 rat cardiomyocytes were labeled by Lifeact;the results showed that the labeling for HUVECs’microfilaments labeling was not obvious compared to those of NRK and H9C2 cells.We believe that HUVECs is a difficult to be transfected cell line,and the transient transfection is not suitable for the Lifeact’s labeling of HUVECs microfilaments,we are seeking lentivirus-mediated stable transfection to solve this problem.In summary,the advanced piezoelectric double resonator sensor technique proposed in this study can be used to quantitatively measure the stress or traction force of HUVECs applied to the surface of the sensor.In combination with fluorescence microscope,the mechanical properties and morphological changes of HUVECs were simultaneously measured.In particular,the changes in cell traction or contractile force and viscoelasticity of HUVECs under the treatments of endothelium barrier agonist thrombin and antagonist y-27632 were simultaneously measured for the first time using the advanced piezoelectric double resonator technique;the results indicated that thrombin increased the cells’contractile force and made the cells stiffer,and y-27632 decreased the cells’contractile force and made the cells softer.The advanced piezoelectric double resonator technique established in this study can be used as an ideal tool for studying the mechanical damage and protection mechanisms of vascular endothelial cells and applied for the evaluation of drugs affecting endothelial barrier and cytotoxicity test.