| Object: To compare the effects of intermittent and constant high insulin media on apoptosis and the production of NO in cultured human umbilical endothelial cells line (HUECV304) so as to explore the mechanism of intermittent hyperinsulinemia on diabetic vascular complication.Methods: Human umbilical vein endothelial cells (EC) line were exposed for 72 hours to 0 nmol/L insulin (control), 1 nmol/L insulin (nomal insulin ), 100 nmol/L insulin (constant high insulin) and 1nmol/L alternating with 100nmol/L insulin per 8 hours (intermittent high insulin) respectively. The cell apoptosis were detected by the method of acridine orange(AO)/ethdium bromide(EB) staining and flow cytometry.The eNOS mRNA expression was determined by RT-PCR.The protein expression of eNOS was detected by western immunoblotting and immunocytochemistry.The production of NO was measured with nitrate reduction test.Results: 1 The effects of insulin on endothelial cell apoptosisAfter cells were treated with various concentration of insulin for 72 hours, by the methodof acridine orange(AO)/ethdium bromide(EB) staining the apoptotic cells increased in bothconstant high insulin group and intermittent high insulin group,which were 1.53-folds(P<0.001) and 1.75 (P<0.001)-folds of control group respectively,and the latter increasedmore significantly (P<0.001) . However the apoptotic cells decreased in nomal insulingroup.by the method of AO-EB fluorescence,which were 81% of control group(P<0.001) .The results by the method of flow cytometry were almost consonant to that ofAO-EB.2 The effects of insulin on the mRNA transcription of eNOS, protein expressions of eNOS and the production of NO(1), In cultured HUECVs, exposure to normal insulin,constant high insulin and intermittent high insulin for 72 hours, caused a marked increase in the mRNA expression of eNOS. Compared with cells exposed to control media,the expressions of eNOS mRNA were... |
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