糖基化终产物引起晚期内皮祖细胞功能障碍

Advanced Glycation End Products Impair Function of Endothelial Progenitor Cells

目的观察与糖尿病患者血清浓度类似的糖基化终产物修饰的牛血清白蛋白对体外培养脐血来源晚期内皮祖细胞功能的影响并探讨可能机制。方法密度梯度离心法分离脐血中单个核细胞,用差速贴壁法分离、培养晚期内皮祖细胞。流式细胞术、免疫细胞化学染色及荧光标记的乙酰化低密度脂蛋白、植物凝集素被用于鉴定培养的细胞。将晚期糖基化修饰的牛血清白蛋白与晚期内皮祖细胞共同培养24h后,利用噻唑蓝法检测细胞的增殖能力,AnnexinV/PI双染法流式细胞术检测细胞凋亡;Boyden小室法检测血管内皮生长因子趋化的细胞迁移;ECMatirx-gel检测形成毛细血管样网状结构的能力。利用逆转录聚合酶链反应、蛋白免疫印迹法测定糖基化终产物受体mRNA和蛋白表达水平。结果脐血单个核细胞在体外培养过程中先后出现两种细胞:第5～7天出现集落样生长细胞,扩增不明显,存在14天左右即消失,这类细胞被称为"早期内皮祖细胞";10～15天时,逐渐出现20～50个细胞组成的细胞簇,1～3天即可形成大于500个细胞簇,细胞呈铺路石样,可传代,大于95%的细胞免疫表型为CD45-/CD146+/CD105+,表达内皮细胞特有的vWF因子,可摄取乙酰化低密度脂蛋白并可与荆豆凝集素1结合,这类细胞被称为"晚期内皮祖细胞"。50～400mg/L晚期糖基化修饰的牛血清白蛋白与晚期内皮祖细胞共培养24h后,与对照组相比,细胞的增殖能力未见明显变化(P>0.05)。当晚期糖基化修饰的牛血清白蛋白浓度大于100mg/L时,与对照组相比,晚期内皮祖细胞的凋亡增加(P<0.05)、血管内皮生长因子趋化的迁移以及在ECMatirx-gel上形成新生血管的能力下降(P<0.05),糖基化终产物受体mRNA和蛋白表达均增加(P<0.05)。结论糖基化终产物通过促进凋亡、抑制迁移及体外形成新生血管的能力引起晚期内皮祖细胞功能障碍,这些影响可能与糖基化终产物上调晚期内皮祖细胞上糖基化终产物受体表达有关。

Aim To investigate effects of advanced glycation end products（AGE）modified albumin（AGE-albumin）on functions of late endothelial progenitor cell（EPC）derived from human umbilical cord blood and its mechanisms.Methods Mononuclear cells from human umbilical cord blood were cultured by using EGM-2-MV（Clonetics）.Expression of CD45,CD146,and CD105 were detected by fluorescence-activated cell sorter analysis（FACS）,vWF was detected by immunocytochemistry.Uptake of acetylated low density lipoprotein（ac-LDL）and binding to Ulex European Agglatinin（UEA-1）were used for fluorescent labeling of EPC.Late EPC were incubated with different concentrations of AGE-albumin which were similar to the diabetic serum for 24 h.MTT was used for EPC proliferation assay.To measure the apoptosis,Annexin V＋/PI-cells were detected by FACS analysis.Boyden chamber assay was used to detect the migration by vascular endothelial growth factor（VEGF）.Tube formation on ECMatrix-gel was performed to assess the capacity for vasculogenesis.The mRNA and protein expression of AGE receptor（RAGE）were evaluated by reverse-transcriptase polymerase chain reaction（RT-PCR）and Western blotting,respectively.Results There were two types of cells appeared in the culture system.After 3 to 5 d,attached cells appeared and appeared to be clusters.Their number increased for 2 weeks.Thereafter,they did not replicate in vitro and gradually disappeared.We observed another population of cells with different morphology and growth pattern.These appeared in 10 to 15 d after plating,withsmooth cytoplasmic outline and were firmly attached to the plate and showed cobblestone appearance,named late EPC.More than 95% of late EPC were CD45-/CD146＋/CD105＋and vWF＋,took-up ac-LDLand showed UEA-1 binding affin-ity.AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation,but did not affect proliferation,of late EPC.High AGE-albumin increased RAGE mRNA and protein expression.Conclusion These results suggest that AGE-albumin make impaired function of late EPC,through up-regulation of RAGE in these cells.