Abstract: PURPOSE: To establish a cell model of human vascular endothelial hypoxia/reoxygenation injury, as an in vitro model of tissue flap ischemia-reperfusion injury (IRI). Methods: Primary human umbilical vein endothelial cells (HUVECs) were cultured to the 5th generation, then divided into 4 groups randomly according to the time of exposing in hypoxia condition as 3h/2h group, 6h/2h group, 9h/2h group and control group. The cells of hypoxia groups were placed in low-glucose and serum-free DMEM and submitted to hypoxia condition for 3, 6 and 9 h respectively,then cultured in normoxia condition with original complete medium for 2 h as reoxygenation. After reoxygenation, morphological features, structures and numbers were observed, the rate of surviving cells was determined by Trypan blue assay, and the cell viability was measured by CCK-8 assay, the rate of apoptotic cells was detected by flow cytometry with Annexin V-PI assay. The data were analyzed with SPSS 19.0 software package. Results: Compared with the control group,the morphology of HUVECs were obviously damaged in HRI groups,with increasing time of hypoxia,the damage of cells gradually aggravated. In Trypan blue and CCK - 8 assays, the rate of surviving cells and the cell viability decreased in HRI groups, with the increase of hypoxia time, the rate of surviving cells and the cell viability decreased significantly (P<0.05). Annexin V-PI assay showed that the early and late apoptotic rate of cells in HRI groups were significantly higher compared with the control group; and with the increase of hypoxia time,the early and late apoptotic rate increased gradually(P<0.05). Conclusions: The reliable cell model of human vascular endothelial hypoxia/reoxygenation injury was established successfully with cultured HUVECs, which provides a basis for in vitro studies of tissue flap ischemia-reperfusion injury.

Key words: Vascular endothelial cells, Hypoxia/reoxygenation injury model, Tissue flap, Ischemia reperfusion injury