The Effects Of Substrate Stiffness On Cells About Wound Repair

In wound repair, the first event is the migration of epidermal cells over the injured dermis. New blood vessels then form by endothelial cell, and the sprouts of capillaries associated with fibroblasts and macrophages replace the fibrin matrix with granulation tissue. The epidermal cells, endothelial cells and fibroblasts that are behind the leading edge proliferate and mature and, finally, restore the barrier function of the skin.The substrate stiffness changed in wound repair and the mechanical properties can regulate proliferation, migration and differentiation. It is value to study the effect of substrat stiffness to cells for wound repair. We use human adult low calcium high temperature cell line (HaCat), human unbilical vein endothelial cell (Huvec), fiboblast as seeded cells. Incubate three kinds of cells on silica substrate with different Young's modulus of elasticity to find the effect of substrate stiffness. And demonstration molecule mechanism about substrate stiffness to give evidence for wound repair.Major contents and results in this thesis:(1)Recongnize changes of substrate stiffness in wound repair. The concentration of curing agent in basic was varied to control the elastic modulus of silica subastate. And we ues scanning electron microscope to observation the substrate surface topography. The results show that we ues 16kpa, 20kpa, 200kpa to simulate substrate stiffness of normal skin, wound granulation tissue in seventh day, late granulation tissue. The surface topographys of different concentrate on silica substrate were not difference.(2)Incubate HaCat, Huvec and fiboblast on silica substrate with different Young's modulus of elasticity. Use cell growth curve, MTT method, cell cycle detect for proliferation and scratch test detect for migration. HaCat, Huvec and fiboblast proliferation were favored on the stiffer surface and them migration also selected stiffer surface. The HaCat and fiboblast are more sensitive to stiffness change than Huvec.(3)SiIntegrinβ1 recombinant vector was transciently transfected into HaCaT cells and fiboblasts , and the changes in the expression of integrinβ1 protein was determined with western blotting; Then the transfected cells undergone hycromycin selection constantly until mono-clone has formed. Expression of integrinβ1 protein was supressed by the transfection of siIntegrinβ1 vector.(4)Incubate siIntegrinβ1 HaCat cells and siIntegrinβ1 fiboblast on high stiffness silica substrate and use HaCat cells and fiboblast as control. Use cell growth curve, MTT method, cell cycle detect for proliferation and scratch test detect for migration. SiIntegrinβ1 HaCat cells and siIntegrinβ1 fiboblasts proliferation activity were lower than HaCat cells and fiboblasts on the high stiffer surface and them migration also more slow than normal cells.