The Effect Of Natural Hirudin On Human Microvascular Endothelial Cells In 3D Culture

Object To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells(HMVECs)and its preliminary mechanism of inducing vascular regeneration.Methods three-dimensional culture model of human microvascular endothelial cells were established in vitro,respectively with low medium and high concentrations(1ATU/ml,4ATU/ml,7ATU/ml of the natural hirudin intervention of the model,and a conventional three-dimensional culture as the control group;the CCK-8(Cell Counting Kit 8)method to detect the levels of cell proliferation,tube formation assay to test the angiogenesis of natural hirudin,cell immunofluorescence to measure the expression level of VEGF and Notch1.Result The observation of cells in three-dimensional culture shows that HMVEC attached to Matrigel gel well,and the cells formed tube structure completely after 24 h.The CCK-8 test detects A value of 1,4 ATU/ml group were higher than that of control group in each time(P < 0.05),and A value of4ATU/ml group was the highest;However,A value of 7ATU/ml group wassignificantly lower than that of 1 ? 4ATU/ml and control group.The tube formation assay shows that the tube structure number of 1,4 ATU/ml group was more than that of 7ATU/ml and control group,and 4 ATU/ml was better than1ATU/ml(P<0.05);But there was no statictically difference between 7ATU/ml group and control group(P>0.05).The Immunofluorescence staining shows that compared with control group,the Notch1 expression of 1ATU/ml,4ATU/ml was higher and the expression of 7ATU/ml was lower;And there was statistically difference between 4ATU/ml group and control group.The VEGF expression of 1,4,7ATU/ml was higher than the expression of control group(P<0.05);4 ATU/ml group is the best(P<0.05).Conclusion Hirudin can promote angiogenesis at low and medium concentrations,but suppress vascular regeneration at high concentrations;Its mechanism can be related to the VEGF-Notch signal pathway.