同型半胱氨酸对外周血内皮祖细胞数量和功能的影响

Effects of homocysteine on number and activity of endothelial progenitor cells from peripheral blood

目的:观察同型半胱氨酸(Hcy)对外周血内皮祖细胞(EPCs)的影响。方法:密度梯度离心法获取外周血单个核细胞,将其接种在人纤维连接蛋白包被的培养板,培养7d后,收集贴壁细胞,加入不同浓度Hcy(10μmol/L、50μmol/L、100μmol/L和200μmol/L)干预一定时间(6h、12h、24h和48h)。多波长激光共聚焦显微镜鉴定FITC-UEA-Ⅰ和DiI-acLDL双染色阳性细胞为正在分化的EPCs,流式细胞仪检测其表面标志进一步鉴定EPCs,倒置荧光显微镜下计数。采用MTT比色法、改良的Boyden小室、粘附能力测定实验和体外血管生成试剂盒分别观察EPCs的增殖能力、迁移能力、粘附能力和体外血管生成能力。结果:Hcy呈量效和时效地减少EPCs数量,200μmol/LHcy作用24h后EPCs数量明显少于对照组(35.7±6.7vs62.5±10.6,P<0.01),并损害EPCs增殖(0.531±0.061vs0.328±0.055,P<0.01)、迁移(26.3±6.4vs6.4±3.7,P<0.01)、贴壁(33.1±8.1vs17.4±7.5,P<0.01)、和体外血管生成(25.4±9.1vs10.4±4.7,P<0.01)。结论:Hcy不但减少EPCs数量并损害其功能。

AIM: To investigate whether homocysteine (Hcy) has influences on endothelial progenitor cell (EPCs) number and activity from peripheral blood. METHODS: Total mononuclear cells (MNCs) were plated on fibronectin-coated culture dishes and cultured for 7 days, and then attached cells were stimulated with Hcy or vehicle control for 6 h, 12 h, 24 h and 48 h. The adhesion, proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed, respectively. RESULTS: Incubation of isolated human MNCs with Hcy dose and time-dependently decreased the number of EPCs with maximum at 200 (μmol/L) for 24 hours (35.7±6.7 vs 62.5±10.6, P<0.01). In addition, Hcy impaired EPC proliferative (0.531±0.061 vs 0.328±0.055, P<0.05), migratory (26.3±6.4 vs 6.4±3.7, P<0.01), adhesive (33.1±8.1 vs 17.4±7.5, P<0.01) and vasculogenesis capacity (25.4±9.1 vs 10.4±4.7, P<0.01) in a dose and time-dependent manner. CONCLUSION: It is suggested that Hcy may result in the reduction of EPCs and decrease EPC functional activity.