关键词: 大鼠, 内皮细胞, 氧化应激, 损伤, 凋亡, 模型

Abstract: Objective: To establish a model of oxidative stress injury in cultured rat aortic endothelial cells, and to provide a basis for the research of cell injury and apoptosis. Methods: The rats were decapitated to get the aorta in thoracic operation under aseptic conditions. By subculture after tissue block culture method to get sufficient aortic endothelial cells, cultured in 96-well plates or grow on cover glass for the following test. Without H₂O₂ group as a control group, with different doses of H₂O₂ (100,200,300,400,500 μmol/L) treated endothelialcells in 12 h to screen the optimal dose. Based on the results, with the same dose of H₂O₂ (100 or 200 μmol/L) acted on endothelial cells respectively in different time (3, 6, 9, 12 and 24 h) to screen the optimal duration. Each group was made in sextuplicate. The establishment of the model was evaluated by immunofluorescence,cell viability testing, biochemical indicators detection (lactate dehydrogenase(LDH), nitric oxide(NO), malondialdehyde(MDA), superoxidedismutase(SOD))and apoptosis index testing. Results: Endothelial cells were cultured successfully and verified by immunofluorescence staining of intracellular antigen Ⅷ collagen. With the increase of H₂O₂ doses at the same action time 12 h, the cell viability was significantly decreased (77.63%±5.20% to 40.90%±2.10%). The same dose(100 μmol/L group and 200 μmol/L group)with the action time increasing, the cellviability was significantly decreased (100 μmol/L group was 86.83%±12.11% to 44.26%±5.70%, 200 μmol/L group was 78.28%±11.98% to 34.45%±5.87%). At dose of H₂O₂ was 100 μmol/L and treated in 3,6,9,12 and 24 h, LDH-L and MDA were significantly increased after 9 h while NO and SOD were significantly decreased. In H₂O₂ dose of 100 μmol/L and action time 12 h, flow cytometry showed endothelial cellapoptosis rate was 16.92%±2.37%, significantly higher than the control group of 2.68%±0.47%(P<0.01); TUNEL detected endothelial cell apoptosis index was17.65%±2.36%, which was significantly higher than that in the control group of 3.23%±0.57%(P<0.01). Conclusion: The method was successfullyestablished a model of oxidative stress injury in cultured rat aortic endothelial cells, explore the moderate conditions that induced cells injury and apoptosis which could be a basis for the research.

Key words: rats, endothelial cells, oxidative stress, injury, apoptosis, model