血红素氧合酶1基因对高血糖和波动性高血糖诱导血管内皮细胞凋亡的影响

摘要

Objective To investigate the effect of heme oxygenase-1 (HO-1) on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by constant high blood glucose and intermittent high blood glucose.Methods Untransfected HUVECs and transfected HUVECs were exposed for 7 days to 5.5 mmol/L lD-glucose (normal glucose),20.0 mmol/L D-glucose (constant high blood glucose) and 5.5 mmol/L alternating with 20.0 mmol/L D-glucose every 24 hours (intermittent high blood glucose),respectively.The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Cell apoptosis was detected by acridine orange (AO)/ethdium bromide (EB) staining.The mRNA expression of bax and bcl-2 was evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis.Results Compared with untransfected HUVECs,the expression level of HO-1 mRNA was increased significantly in transfected HUVECs [(0.91 ± 0.29) vs (0.43 ± 0.14),P ＜ 0.05].Transfected HUVECs showed higher viability (108 ± 27,87 ± 9)than untransfected HUVECs(P ＜ 0.05).Transfected HUVECs had a lower apoptotic cells(35.0,48.0) than untransfected HUVECs(P ＜0.05).Transfected HUVECs had higher mRNA expression of bcl-2 compared with untransfected HUVECs.Conclusion The up-regulation of gene HO-1 protect HUVECs from apoptosis induced by constant high glucose and intermittent high glucose is associated with the up-regulation of bcl-2 expression.%目的 探讨血红素氧合酶1对高血糖和波动性高血糖诱导内皮细胞凋亡的影响.方法 以未转染及转染血红素氧合酶1基因的人脐静脉内皮细胞为研究对象,实验分正常葡萄糖组、波动性高血糖组、正常葡萄糖/血红素氧合酶1组、稳定性高血糖/血红素氧合酶1组及波动性高血糖/血红素氧合酶1组.细胞分别置于含正常浓度葡萄糖(5.5 mmol/L)、稳定性高浓度葡萄糖(20.0 mmol/L)和波动性高浓度葡萄糖(5.5或20.0mmol/L)的培养基,培养7d后用噻唑蓝(MTT)方法检测细胞存活率,吖啶橙/溴乙啶(AO/EB)染色法检测细胞凋亡,逆转录-聚合酶链反应(RT-PCR)检测凋亡相关基因bax、bcl-2 mRNA的表达.结果 转染内皮细胞血红素氧合酶1 mRNA表达量明显高于未转染内皮细胞[(0.91 ±0.29)比(0.43 ±0.14),P＜0.05].正常葡萄糖组、正常葡萄糖/血红素氧合酶1组、稳定性高血糖组、稳定性高血糖/血红素氧合酶1组、波动性高血糖组、波动性高血糖/血红素氧合酶1组细胞存活率分别为(100 ±22)％、(1 10 ± 19)％、(79 ± 11)％、(108 ±27)％、(59±13)％、(87 ± 9)％,凋亡细胞数分别为(33 ± 7)、(32 ±7)、(53 ±5)、(35 ±7)、(59 ±7)、(48 ±7).稳定性高血糖组和波动性高血糖组细胞存活率较正常葡萄糖组明显下降(P＜0.05或P＜0.01),且波动性高血糖组细胞存活率下降更为明显(P＜0.05);稳定性高血糖/血红素氧合酶组及波动足性高血糖/血红素氧合酶组细胞存活率较稳定性高血糖组及波动性高血糖组均提高(P＜0.05).正常葡萄糖组、正常葡萄糖/血红素氧合酶1组、稳定性高血糖组、稳定性高血糖/血红素氧合酶1组、波动性高血糖组、波动性高血糖/血红素氧合酶1组bax/β-actin表达分别为(1.27 ±0.32)、(1.26 ±0.35)、(1.13 ±0.32)、(1.13 ±0.39)、(1.32 ±0.45)、(1.31 ±0.41),bcl-2/β-actin表达分别为(1.35 ±0.39)、(1.31 ±0.34)、(0.77 ±0.35)、(1.34 ±0.46)、(0.20 ±0.12)、(0.82 ±0.23).稳定性高血糖组与波动性高血糖组bcl-2的表达比正常葡萄糖组明显减弱(P＜0.05或P＜0.01),与稳定性高血糖组和波动性高血糖组相比,稳定性高血糖/血红素氧合酶1组、波动性高血糖/血红素氧合酶1组bcl-2 mRNA表达增强(P＜0.01).结论 血红素氧合酶1基因的表达上调可减轻高血糖和波动性高血糖引起的血管内皮细胞凋亡,其机制与bcl-2的表达上调有关.