摘要
目的观察雷帕霉素对外周血内皮祖细胞(EPCs)数量与功能的影响。方法采用密度梯度离心法从外周血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上,培养7天后收集贴壁细胞,加入不同浓度(1·0、2·0、5·0μg/ml)雷帕霉素分别培养6、12、24、48h。激光共聚焦显微镜鉴定,FITC-UEA-I和Dil-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以进一步鉴定EPCs,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来观察EPCs的增殖能力、迁移能力、黏附能力和体外血管形成能力。结果EPCs数量随雷帕霉素浓度与作用时间增加而减少,其增殖能力、迁移能力、黏附能力和体外血管形成能力亦随雷帕霉素浓度与作用时间增加而降低。结论雷帕霉素降低EPCs的数量、增殖能力、迁移能力、黏附能力和体外血管形成能力,并呈浓度和时间依赖性。
Objective To investigate the effects of rapamycin on the number and function of peripheral blood endothelial progenitor cells （EPCs）. Methods 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-day culture, adherent cells were treated with raparnycin in a series of final concentrations of 1.0, 2. 0, 5.0μg/rnl for 6, 12, 24, and 48h. EPCs were identified as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining as demonstrated under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of VEGFR-2, AC133 and CD34 with flow cytometry. EPCs proliferation and migration were assayed with MTT assay and modified Boyden chamber assay, respectively. EPCs adhesion assay was performed by replating them on fibronectin-coated dishes, and then adherent cells were counted. In vitro vasculogenesis activity was assayed by in vitro vasculogenesis kit. Results Incubation of isolated human MNCs with rapamycin resulted in a decrease in the number of EPCs, and rapamycin also decreased EPCs proliferative, migratory, adhesive and in vitro vasculogenesis capacity in both concentration and time dependent manners. Conclusion Raparnycin decreases the number, proliferative, migratory, adhesive and in vitro vasculogenesis capacity of EPCs.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2006年第4期316-318,共3页
Medical Journal of Chinese People's Liberation Army
关键词
雷帕霉素
内皮祖细胞
细胞培养
raparnycin
endothelial progenitor cells
cell culture
