晚期糖基化终产物对内皮祖细胞功能及成骨分化的影响  

Effects of Advanced Glycation End Products on Function and Osteogenic Differentiation of Human Endothelial Progenitor Cells

作　　者：汪沁沁[1] 岳温恒 伍锋[1] 贺治青[1] 梁春[1] 吴宗贵[1] 

机构地区：[1]中国人民解放军第二军医大学附属长征医院,上海200003

出　　处：《中西医结合心脑血管病杂志》2016年第3期266-270,共5页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：国家自然科学基金重点项目(No.81130065);国家自然科学基金青年项目(No.81270405)

摘　　要：目的观察糖尿病机体主要的晚期糖基化终产物亚型-Nε羧甲基赖氨酸修饰白蛋白(CMLs)对内皮祖细胞(EPCs)功能及成骨分化作用的影响。方法采用密度梯度离心法从外周单个核细胞中分离、培养得到EPCs,通过观察细胞形态和流式细胞仪检测细胞表面标记鉴定。进一步采用CCK-8增殖试剂盒、Annexin V-FITC/PI流式凋亡试剂盒、transwell小室迁移实验分别观察空白对照组、BSA对照组(100μg/mL)、不同浓度CML-BSA组(20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL),共7组干预液对EPCs增殖、凋亡、迁移功能的影响。并检测成骨相关钙化基因BMP-2、ALP、RUNX-2和OCN的表达量,观察其对EPCs成骨分化能力的影响。结果与BSA对照组相比,CML-BSA能显著抑制EPCs增殖和迁移的能力,增加凋亡,促进其成骨分化(P<0.05),其中80μg/mL浓度变化最明显(P<0.05);与BSA对照组相比,CML-BSA能显著增加成骨相关钙化基因BMP-2、ALP、RUNX-2和OCN的表达量,呈现浓度依赖性递增,80μg/mL浓度变化最大(P<0.05),100μg/mL浓度呈现下降趋势(P<0.05)。结论 CMLs能抑制EPCs的增殖和迁移能力,增加其凋亡,显著促进EPCs成骨分化。深入研究有助于探索糖尿病血管钙化机制,为临床干预提供新靶点。Objective To investigate the effects of Nε-（carboxymethyl）Lysine albumin（CMLs）,aprimary advanced glycation end products isoform in diabetic body,on function and osteogenic differentiation of human endothelial progenitor cells（EPCs）.MethodsEPCs were isolated and cultured by Ficoll density gradient centrifugation from human mononuclear cells,then were identified to observe cultured cells’ morphology and detected cell surface marker by flow cytometry.The cells were exposed to 7 different interventions respectively for 24 hours,including PBS,100μg/mL BSA,and CML-BSA（20,40,60,80,100μg/mL）.The proliferation capability of such cells was evaluated using CCK-8 assay,migration ability was explored by transwell assay,and apoptotic rates were investigated by Annexin V-FITC/PI FCM kits.Results Compared with the BSA group,proliferation and migration capability of EPCs were inhibited by stimuli with CML-BSA（n =6,P 〈0.05）,but apoptosis of such cells were promoted,and the concentration of CML-BSA（80μg/mL）was the most effective one.Compared with the BSA group,the expression levels of osteogenesis-related genes,like bone morphogenetic protein 2（BMP-2）,alkaline phosphatase（ALP）,runt-related transcriptional factor 2（RUNX-2）and osteocalcin（OCN）,were increasing gradually by CML-BSA treatment,and it shown the concentration-dependent.The concentration of CML-BSA（80μg/mL）was the most effective one,but the effect of concentration of CML-BSA（100μg/mL）went down.Conclusion CMLs could significantly reduce proliferation,migration,capability,but promote apoptosis and the expression levels of osteogenesis-related genes of EPCs.It could be helpful to explore the mechanism of diabetic vascular calcification,and provide new drug targets for clinical intervention.

关 键 词：糖尿病 内皮祖细胞 晚期糖基化终产物 成骨分化 

分 类 号：R587.2[医药卫生—内分泌]