Studies Of Biomechanics During Differentiation Of Human Amniotic Mesenchymal Stem Cells And Inhibition Effect Of Gold Nanoparticles On Angiogenesis

This dissertation includes two parts:(1) the changes of mechanical properties during lipoblast and osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs) by atomic force microscopy (AFM), (2) The inhibition effect of gold nanoparticles on angiogenesis.Human amniotic mesenchymal stem cells are becoming an important source of cells for regenerative medicine given with apparent advantages of accessibility, renewal capacity and multipotentiality. Atomic force microscopy (AFM), Laser Scanning Confocal Microscope (LSCM) and Flow Cytometry were used to study the changes of mechanical properties during osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs). It is found that as osteogenic differentiation of hAFSCs progressed, more and more stress fibers were replaced by a thinner actin network. The mechanical properties of human amniotic mesenchymal stem cells (hAMSCs), such as the average Young's modulus, were determined by atomic force microscopy (3.97±0.53 kPa for hAMSCs vs.1.52±0.63 kPa for fully differentiated osteoblasts). The cytoskeleton arrangements may result in decreasing Young's modulus. Furthermore, ultrastructures, nanostructural details on the surface of cell, were visualized by atomic force microscopy (AFM). It was clearly shown that surface of osteoblasts were covered by mineralized particles, and the histogram of particles sizes showed that most of the particles on the surface of osteoblasts distributed from 200 to 400 nm indiameter, while the diameter of (hAMSCs), particles ranged from 100 to 200 nm. Moreover, when hAMSCs were differentiated into lipoblast, the visco-elasticity increased, the stiffness decreased.In the second part, gold nanoparticles can specifically block the interaction of vascular endothelial growth factor165 (VEGF165) with vascular endothelial growth factor receptor (VEGFR-2), which makes the gold nanoparticle has the anti-angiogenesis and anti-cancer effect. Force spectroscopy of atomic force microscopy (AFM) was applied to study the banding force of VEGF165 -VEGFR-2. When human Umbilical Vein Endothelial Cells (hUVECs) were exposed to gold nanoparticles solution for 24 h, AFM high-resolution image of plasmodesmata showed that the plasmodesmata became significantly thinner then untreated cells. Furthermore, on the molecular level, force spectroscopy was also used to analyze the banding force of VEGF165 -VEGFR-2(119.35±44.3 pN). When VEGF165 was added into growth media the binding force between VEGF165 and VEGFR-2 was dropped into 31.6±11.0 pN, which means that the banding force between VEGF165 and VEGFR-2 is specificity. However, when the blocking solution was replaced by Au-VEGF165 conjunction, the binding force was up to 108.3±38.1 pN, It means that the interaction between gold nanoparticles and VEGF165 primarily occurs through the heparin binding domain of the protein thus inhibit the interaction between VEGF165 -VEGFR-2. The results mentioned above revealed that the mechanism of the anti-angiogenic properties of gold nanoparticles is:the interaction between gold nanoparticles and VEGF165 primarily occurs through the heparin binding domain of the protein, which makes the gold nanoparticles blocking the binding of VEGF165 to its receptors. Thus, gold nanoparticles have the antiangiogenic effect.