晚期糖基化终末产物调节氧化应激对内皮祖细胞生物学功能的影响

目的 观察晚期糖基化终末产物对体外培养的内皮祖细胞氧化应激状况的改变及对内皮祖细胞生物学功能的影响,探讨晚期糖基化终末产物参与动脉粥样硬化发生发展的可能作用位点.方法 使用密度梯度离心法从骨髓获取单个核细胞并进行体外培养.对贴壁细胞进行细胞化学分析,以激光共聚焦显微镜鉴定FITC标记荆豆凝血素I和DiI-标记的乙酰化低密度脂蛋白双染色阳性细胞为正在分化的内皮祖细胞.在内皮祖细胞中加入不同浓度晚期糖基化终末产物,测定晚期糖基化终末产物作用24 h后内皮祖细胞内活性氧、超氧化物岐化酶、谷胱甘肽过氧化物酶含量,同时测定内皮祖细胞迁移、黏附和增殖能力.结果 随晚期糖基化终末产物作用浓度升高(0、50、100和200 mg/L),内皮祖细胞内活性氧含量明显增加(分别为2.78±0.12、5.98±0.11、6.42±0.12和7.39±0.09,P<0.01),超氧化物岐化酶(分别为100%、81.2%±3.1%、71.0%±3.1%和44.2%±3.3%,P<0.01)、谷胱甘肽过氧化物酶(分别为100%、80.4%±3.6%、68.6%±3.7%和40.6%±4.2%,P<0.01)等抗氧化酶mR-NA含量减少,活性减退(P<0.01),内皮祖细胞生物学功能明显减退(P<0.01).结论 晚期糖基化终末产物促进内皮祖细胞内活性氧生成,减少抗氧化酶的合成,增强氧化应激,破坏细胞内环境稳定性,使细胞生物学功能受损.并且这种变化与晚期糖基化终末产物浓度成正相关.

Advanced Glycation Endproducts Alters Functions in Endothelial Progenitor Cells Through Oxidant Stress

Aim To investigate the effects of advanced glycation endproducts (AGE) on oxidative stress in bone-marrow derived endothelial progenitor cells (EPC) and the relationship of EPC functions.Methods Mononu-clear cells (MNC) were isolated from rat bone marrow by Ficoll density gradient centrifugation and cultured for 7 days,EPC were identified as adherent cells double positive stained for FITC-UEA-Ⅰand DiI-acLDL under laser confocal immunofluencemicroscope.EPC were cultured in the absence or presence of AGE (50, 100 and 200 mg/L).Reactivated ox-ygen species (ROS) were analyzed using the ROS assay kit.A spectrophotometer was used to assess superoxide dis-mutase (SOD) and glutathione peroxidase(GSH-Px) expression, and the PCR was also used to determine the mRNA ex-pression.A modified Boyden's chamberwas used to assess the migration of EPC and the number of recultured EPC was counted tomeasure the adhesiveness function.A spectrophotometer was used to determine the proliferation function.Results Co-culturing with AGE increased ROS production, decreased antioxidase and induced the proliferation, migra-tion and adhesion of EPC inhibition in a dose dependentmanner.Conclusion AGE increase oxidative stress andme-diate impairment of progenitor cell function in a dose dependentmanner, which indicates a new pathophysiologicalm echa-nism of disturbed vascular adaptation in atheroscleros is and suggests that lower levels of AGE might improve the success of progenitor cell therapy.