The Effects Of Substrate Stiffness On The Biological Behaviour Of Human Dermal Microvascular Endothelial Cells

Objective:In vivo,the normal physiological function of vascular endothelial cells are regulated by vascular basement wall stress and shear stress in the mechanical stimulation,and most of the vascular endothelial cells cultured in vitro is in the traditional organization(tissue culture plate,TCP),and the vascular endothelial cell body located in the vascular wall of the hardness some differences may affect the normal physiological function of cells.In this study,starting from the angle of biomechanics,simulated vascular physiological state of vascular stiffness with different hardness of polyacrylamide hydrogel,the traditional tissue culture plate as control,to explore the different substrate hardness on human dermal microvascular endothelial cells(Human Dermal Microvascular Endothelial Cells,HDMEC)influence the biological behavior of the extension and proliferation.On this basis,the relevant gap occurrence and development by detecting the connexin and atherosclerosis and endothelial specific cell glycocalyx,differences between the comparison of different substrate stiffness,in order to obtain a suitable substrate hardness in vitro.Methods:In this study HDMEC were cultured in the basal 4k Pa,25 k Pa,hardness of the50 k Pa hydrogel(hydrogel,GEL)plastic plate culture and traditional organization(tissue culture plate,TCP),microfilament cytoskeleton protein by immunofluorescence labeled HDMEC to detect cell proliferation level.Western(blot)and real-time fluorescent quantitative PCR were used to detect the expression level of gap junction protein(connexin 40,CX40),and the content of glycocalyx was detected by means of the test kit.Results:The experimental results show that the observed under the fluorescence microscope,with the increase of substrate hardness of HDMEC form is gradually extended,in the TCP cells were spindle shaped cells and the largest area in the hydrogel cells in the round;there is no significant difference between the rate of cell proliferation on the cultured hydrogel,but was significantly lower than that of TCP;the expression level of training in the hydrogel HDMEC CX40 RNA and protein than TCP high,the 4k Pa HDMEC CX40 was the highest;through the detection of cell surface glycocalyx content,cell surface glycocalyx content of hydrogel culture was higher than that of TCP,and the 4k Pa HDMEC cell surface glycocalyx content was the highest.Conclusions:The hydrogel with substrate stiffness of 4k Pa is more suitable for the culture of microvascular endothelial cells in vitro.