Study On The Effect Of AMPK On Hypoxia - Induced Angiogenesis

Objective: To establish a simple and effective method for isolation and culture of humanumbilical vein endothelial cells (HUVEC), and to explore the effect of hypoxia on angiogenesis inHUVEC, which may provide the foundation for the treatment of ischemic diseases.Methods: Primitive culture of HUVECs: the type I collagenase perfusion digestion method wasadopted to isolate umbilical vein endothelial cells, which can further be identified throughimmunofluorescence staining with people first Ⅷ factor related antigen antibody. MTT test wasused to measure the cell activity. The model of hypoxia was induced by CoCl2treatment. Theexpression of p-AMPK/AMPK was detected by western blot. The cultured HUVECs were dividedinto five groups: normal group, hypoxia group (0.2mM CoCl2), hypoxia+Compound C (20μM),hypoxia+C75(10μM), and normal group+VEGF. The migration of HUVECs was measured byscratch-wound assay, and angiogenesis was detected by tube formation assay in vitro.Results: Immunofluorescence staining with people first Ⅷ factor related antigen antibody:thenucleus is stained in blue, cytoplasm with red dye; Which the liquid and the control group (PBS)is no red dye in the cytoplasm. The concentration of CoCl2model is0.2mM, hypoxiaexperimental model is established. AMPK phosphorylation level significantly increased comparedwith the normal control group, hypoxia promoted endothelial cell migration（p<0.05）, promotedtube formation of endothelial cell on the gel（p<0.05）. When was gived AMPK inhibitor,migration and tube formation of endothelial cell and obviously was inhibited by hypoxia.Conclusion: The experiment shows that hypoxia could promote angiogenesis by activation ofp-AMPK, which increases migration of human umbilical vein endothelial cells and tubeformation.