雷帕霉素和紫杉醇对骨髓内皮祖细胞数量及功能的影响

摘要

目的:研究雷帕霉素和紫杉醇对骨髓内皮祖细胞(endothelial progenitor cells,EPCs)数量及功能的影响.方法:将离心法获得的SD雄性大鼠的骨髓单个核细胞接种于明胶包被的培养板上,培养7 d后收集贴壁细胞,加入不同浓度的雷帕霉素(0.1 μg/mL,1μg/mL和5μg/mL)及紫杉醇(0.1μg/mL,1μg/mL和5μg/mL)培养24 h.在激光共聚焦显微镜下观察细胞,DiI标记的乙酰化低密度脂蛋白(DiI-acLDL)和异硫氰酸荧光素标记的单叶豆凝集素1(FITC-BS-1 Lectin)双染色阳性的细胞为正在分化的EPCs,在倒置荧光显微镜下对其进行计数.然后分别采用四甲基偶氮唑盐 (MTT)比色法、改良的Boyden小室法和黏附能力检测实验来观察EPCs的增殖能力、迁移能力及黏附能力.结果:EPCs数量随着雷帕霉素及紫杉醇浓度的增加而减少,其增殖能力、迁移能力及黏附能力亦随药物浓度的增加而降低,各项指标有统计学意义(P<0.05).相同浓度的雷帕霉素和紫杉醇对EPCs数量及功能的影响无显著差异.结论:雷帕霉素和紫杉醇可降低EPCs的数量及其增殖能力、迁移能力和黏附能力,且两种药物对EPCs的效应呈浓度依赖性.%Objective:To investigate the effects of rapamycin and paclitaxel on the number and activity of bone marrow - derived endothelial progenitor cells(EPCs). Methods: Bone marrow mononuclear cells were isolated from rat bone marrow by centrifu-gation and they were cultured on gelatin - coated dishes. After 7- day culture, adherent cells were treated with rapamycin or paclitaxel in a series of concentrations (0. 1μg/mL, 1μg/mL, and 5 μg/mL) for 24 h. EPCs were identified as adherent cells double positive for DiI - acLDL - uptake and lectin binding by direct fluorescent staining. Proliferation and migration of EPCs were determined using MTT assay and modified version of the Boyden chamber assay, respectively. EPCs adhesion assay was performed by replanting the cells on gelatin- coated dishes, and then adherent cells were counted. Results: Incubation of EPCs with rapamycin or paclitaxel decreased the number of EPCs. Rapamycin and paclitaxel also decreased the proliferative, migratory, and adhensive capacity of EPCs in a concentration - dependent manner(P<0. 05). At the same time, the influence of the same concentrations of rapamycin and paclitaxel on the number and function of EPCs showed no significant differences. Conclusions: Rapamycin and paclitaxel decreased the number, as well as the proliferative, migratory, and adhensive capacity of EPCs.