Chen JunZhu, Wang XingXiang, Zhu JunHui, Shang YunPeng, Guo XiaoGang, Sun Jian
Department of Cardiovascular Diseases, Medical School of Zheijiang University, Zheijiang Province, P.R. China.
J Cardiovasc Pharmacol. 2004 Mar;43(3):347-52. doi: 10.1097/00005344-200403000-00004.
The aim of this study is to investigate whether Ginkgo biloba extract can augment endothelial progenitor cells numbers, and promote the cells' proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. Total mononuclear cells 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 Ginkgo biloba extract (to make a series of final concentrations: 10 mg/L, 25 mg/L, and 50 mg/L) or vehicle control for the respective time points (6 hours, 12 hours, 24 hours, and 48 h). Endothelial progenitor cells were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. Endothelial progenitor cells proliferation, migration, and in vitro vasculogenesis activity were assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. Endothelial progenitor cells adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Incubation of isolated human mononuclear cells with Ginkgo biloba extract dose- and time-dependently increased the number of endothelial progenitor cells, maximum at 25 mg/L, 24 hours (approximately 1-fold increase, P < 0.01). In addition, Ginkgo biloba extract also dose- and time-dependently promoted endothelial progenitor cells proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. The results of the present study defined a novel functional effect of Ginkgo biloba extract: the augmentation of endothelial progenitor cells with enhanced functional activity.
本研究旨在探讨银杏叶提取物是否能增加内皮祖细胞数量，并促进这些细胞的增殖、迁移、黏附及体外血管生成能力。通过Ficoll密度梯度离心法从外周血中分离出总单核细胞，然后将细胞接种于纤连蛋白包被的培养皿上。培养7天后，贴壁细胞分别用银杏叶提取物（制成一系列终浓度：10 mg/L、25 mg/L和50 mg/L）或溶剂对照处理相应时间点（6小时、12小时、24小时和48小时）。在激光扫描共聚焦显微镜下通过直接荧光染色将内皮祖细胞鉴定为对DiLDL摄取和凝集素结合呈双阳性的贴壁细胞。通过流式细胞术检测KDR、VEGFR-2和AC133的表达进一步进行记录。分别用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法、改良Boyden小室法和体外血管生成试剂盒检测内皮祖细胞的增殖、迁移和体外血管生成活性。内皮祖细胞黏附试验通过将细胞重新接种于纤连蛋白包被的培养皿上，然后计数贴壁细胞来进行。用银杏叶提取物孵育分离的人单核细胞，内皮祖细胞数量呈剂量和时间依赖性增加，在25 mg/L、24小时时达到最大值（增加约1倍，P < 0.01）。此外，银杏叶提取物还呈剂量和时间依赖性地促进内皮祖细胞的增殖、迁移、黏附及体外血管生成能力。本研究结果确定了银杏叶提取物的一种新的功能作用：增强内皮祖细胞数量并提高其功能活性。
