| Objective: 1. To culture Human Umbilical Vein Endothelial Cells(HUVEC) invitro. 2. To investigate the effects of high level of glucose and insulin on endothelial cell apoptosis. 3. To detect the expression of Mn-SOD and total activity of SOD in hyperglycemia and hyperinsulinemia state.Methods: 1. The cultured HUVEC were characterized by microscope observationand specific immunofluorescence. 2. The cultured HUVEC were incubated at different concentration of D-glucose and insulin for 72 hours,then EC apoptosis was measured through Capase-Glo?3/7 Reagent and Luminometer. 3. The expression of Mn-SOD were measured through SQ-RT-PCR (semi-quantitative reverse transcription- polymerase chain reaction) and activity of total SOD were detected by SOD Test Kit.Result: 1.ECs (endothelial cell) present cobblestone-shaped and a positiveimmuno- fluorescent reaction,and >95% EC grew well. 2. ECs apoptosis increased notably at high concentration of glucose and/or insulin compared with control group(P<0.05). 3. At the same time, the expression of Mn-SOD decreased markedly in hyperglycemia and/or hyperinsulinemia group(P<0.05). 4. Total SOD activity have not changed like the change of Mn-SOD expression and apoptosis.Conclusion: The study confirmed that hyperglycemia and/or hyperinsulinemia could have harmful impact on HUVEC antioxidative capability.Even in the hyperinsulinemia phase not hyperglycemia state,the HUVEC apoptosis have increased and accelerated atherogenesis.This impact on HUVEC apoptosis andantioxidative capability will continue in the diabetes progression. This experiment provided some information about the relationship of diabetic macrovascular complication and oxidative stress. |
