葛根素对外周血内皮祖细胞数量和功能的影响

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

目的 :观察葛根素对外周血内皮祖细胞的影响。方法 :采用密度梯度离心法从外周血获取单个核细胞 ,将其接种在人纤维连接蛋白包被培养板 ,培养 7d后 ,收集贴壁细胞 ,加入不同浓度葛根素 (分别为 0 .1,0 .5 ,1,3mmol·L-1)培养一定的时间 (6 ,12 ,2 4 ,4 8h)。通过激光共聚焦显微镜鉴定FITC UEA Ⅰ和DiI acLDL双染色阳性细胞被认为是正在分化的EPC ,并在倒置荧光显微镜下计数。然后分别采用MTT比色法、改良的Boyden小室、黏附能力测定实验和体外血管生成试剂盒观察EPC的增殖能力、迁移能力、黏附能力和体外血管生成能力。结果 :葛根素显著增加外周血EPC数量 ,并且EPC数量随葛根素浓度增加及作用时间延长而增加 ,3mmol·L-1浓度葛根素作用 2 4h对EPC数量的影响最为显著 (较对照组增加了 1倍 ,P <0 .0 1)。葛根素也显著改善了外周血EPC的黏附能力、迁移能力、增殖能力和体外血管生成能力。结论

Objective: To investigate whether puerarin can augment endothelial progenitor cells (EPCs) numbers, promote EPC proliferation, migration and adhesion. Method: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with puerarin (to make a series of final concentrations: 0.1, 0.5, 1, 3 mmol·L^(-1)) or vehicle control for the respective time points(6, 12, 24, 48 h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay,modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, then adherent cells were counted. Result: Incubation of isolated human MNCs with puerarin dose increased the number of EPCs, maximum at 3 mmol·L^(-1), 24 hours (approximately 1-fold increase, P<0.01). In addition, puerarin also promoted EPC proliferative, migratory,adhesive and in vitro vasculogenesis capacity. Conclusion: Puerarin can augmentf the number of EPCs with enhanced functional activity.