熊果酸对兔外周血内皮祖细胞数量和功能的影响

Effects of Ursolic Acid on Number and Function of Endothelial Progenitor Cells from Peripheral Blood of Rabbits

目的观察熊果酸对兔外周血内皮祖细胞(EPC)数量和功能的影响并探讨其可能的作用机制。方法以密度梯度离心法分离兔外周血单个核细胞,并接种于纤连蛋白包被的培养板上,予含内皮细胞生长添加剂(ECGS)的M199培养基培养14天,应用免疫荧光染色及流式细胞仪检测、鉴定内皮祖细胞,分别以MTT法、改良的Boyden小室、黏附能力测试、ELISA法观察不同浓度(5,10,50,100mmol/L)熊果酸对培养的EPC增殖、迁移、黏附及分泌功能的影响;同时观察糖皮质激素受体拮抗剂RU486预作用的效果。结果熊果酸可浓度依赖性减少兔EPC的数量,并减弱其增殖、迁移、黏附和分泌能力;RU486可部分逆转熊果酸的上述抑制作用。结论熊果酸可能部分通过糖皮质激素受体的介导影响EPC的数量和功能,而这种抑制作用可能使其在抗动脉粥样硬化和抗再狭窄的应用中受限。

Objective To observe the effects of ursolic acid on number and function of endothelial progenitor cells （EPC）, and to find out its possible mechanism. Methods Total mononuclear cells were isolated from peripheral blood of rabbits by density gradient centrifugation and then plated on fibronectin-coated culture dishes with M199 which contained endothelial cell growth supplements（ECGS）. After 14 days, attached cells which were double positive for Dil-acLDL and FITC-UEA-1 under a laser scanning confocal microscope were characterized as EPCs. They were further confirmed by expression CD34 with flow cytometry. Then EPCs were incubated with a series of concentrations of ursolic acid（5,10,50,100μmol/L） for 24h. In order to explore its possible mechanism, attached cells were pretreated with RU486, a glucocorticoid receptor antagonist, for 30min, then incubated with corresponded concentration of ursolic acid. The functions of EPCs such as proliferation, migration and secretion of VEGF and G-CSF were determined by MTT assay, modified Boyden chamber assay and ELISA. The adhesion assay was performed by replating EPCs on fibronectin-coated dishes. Result Ursolic acid decreased the number and weakened the function of EPCs in a dose-dependent manner. The inhibition effect of ursolic acid was partly blocked by pretreatment of RU486. Conclusion It is suggested that ursolic acid could affect number and function of EPCs partly through glucocorticoid receptor and this inhibition effect perhaps would limit its application in antiatherosclerosis and antirestenosis.