Studying The Interaction Of Biomolecules And The Properties Of Cell Surface Based On Atomic Force Microscopy

Atomic force microscopy(AFM)has been developed as a powerful tool for high resolution imaging and the quantitative force measurement with the nanometer-level resolution and piconewton-level force sensitivity.Based on AFM,the surface morphology of samples,the binding between biomolecules and the nanomechanical property of living cells in near-physiological condition can be characterized.In this dissertation,the following researches were carried out using AFM:1.The interaction between VEGF165(the major form of Vascular endothelial growth factor A)and heparin was explored using atomic force microscopy based single-molecule force spectroscopy(AFM-based SMFS)and molecular dynamics(MD)simulation.The binding forces between VEGF165 and heparin at different loading rates was quantified by AFM-based SMFS.The thermodynamic and kinetic parameters of the dissociation process were also obtained.The most likely binding conformation of VEGF165/heparin complex was visualized using MD simulation.The binding free energy of VEGF165/heparin complex was calculated using the molecular mechanics Poisson-Boltzmann solvent accessible surface area(MM-PBSA)method according to the trajectory of MD simulation.It indicated that hydrophobic interaction and hydrogen bonding plays a positive role in the combination between the two molecules.It offered us a new insight into the binding between VEGF165 and heparin.2.The protective mechanism of panax notoginseng saponins(PNS)against oxidative stress was firstly investigated by studying the cell surface biomechanical properties on the single living human umbilical vascular endothelium cell(HUVEC)based on AFM.PNS could distinctly restrain the reduction of cells young's modulus and the dissociation of cytoskeleton caused by oxidative stress damage.It indicates that improving the stability of cytoskeleton is a significant way for PNS to play a protective role in HUVECs during oxidative damage.This work provides a new idea for exploring the functional mechanism of traditional Chinese medicine at the single cell level,and reveals great potential of the atomic force microscope in studying the drug mechanism.3.The process of epithelial-mesenchymal transformation(EMT)of bladder cancer T24 cells was investigated using AFM by observing the change of morphology,mechanical properties and E-cadherin protein(a kind of tumor marker molecule on the cell surface)of T24 cells during EMT.Results showed that T24 cells had mesenchymal characteristics compared with normal bladder epithelial cells,and TGF-?1 could continuously induce EMT in T24 cells.The cytoskeletal rearrangement and F-actin formation in T24 cells during EMT resulted in changes in cell morphology and mechanical properties.The molecular recognition imaging technology was used to quantitatively characterize the expression charges of E-cadherin on the T24 cell surface during EMT,revealed the downregulation trend of E-cadherin.This work shown us the application prospect of AFM in the characterization of trace amounts of proteins on the cell surface,enhanced our understanding of the EMT process of bladder cancer cells at the single moleculer and single cell level,which was helpful for providing us a deeper understanding of tumor metastasis.