辛伐他汀对大鼠平滑肌祖细胞和内皮祖细胞迁移的影响

Differential effects of simvastatin on migration of rat smooth muscle progenitor cells and endothelial progenitor cells

目的观察辛伐他汀对平滑肌祖细胞(smooth muscle progenitor cell,SPC)和内皮祖细胞(endothelialprogenitor cell,EPC)迁移的影响,筛选新一代包被洗脱支架药物。方法采用密度梯度离心法获取大鼠骨髓单个核细胞,重悬于SPC培养基或EPC培养基,接种在纤维连接素包被培养板,平滑肌肌动蛋白免疫荧光染色鉴定骨髓源性SPC,激光共聚焦显微镜鉴定Dil标记乙酰化低密度脂蛋白(DiI-acLDL)和FITC标记的荆豆凝集素Ⅰ(FITC-UEA-Ⅰ)双染阳性细胞为正在分化的EPC。分别收集培养8 d的SPC和EPC,加入不同浓度辛伐他汀(0,0.01,0.1,1,10μmol/L)培养24 h。采用改良Boyden小室检测SPC和EPC迁移能力。结果辛伐他汀显著抑制SPC迁移,0.01μmol/L辛伐他汀作用24 h,迁移SPC数量减少,0.01μmol/L辛伐他汀组与对照组SPC迁移比较,差异有统计学意义(39±3 vs.44±3,n=5,P<0.05)。与SPC相反,辛伐他汀显著促进EPC迁移,其促进作用随辛伐他汀浓度升高而增加,1.0μmol/L时达最大效应,1.0μmol/L辛伐他汀组与对照组EPC计数比较,差异有统计学意义(37±5 vs.6±3,n=5,P<0.01)。结论辛伐他汀选择性抑制SPC迁移,促进EPC迁移,其双向调节作用呈浓度依赖性,局部应用有促进损伤血管再内皮化和抑制新内膜过度增生的可能。

Objectives To investigate different influences of simvastatin on migration of rat smooth muscle progenitor cells（SPC） and endothelial progenitor cells（EPC）,and identification of compounds that differentially inhibit SPC and EPC migration for substantial clinical usefulness.Methods Total mononuclear cells（MNCs） were isolated from marrow of rats by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.SPC outgrew from the culture of MNC in SPC medium（the presence of platelet-derived growth factor-BB and basic fibroblast growth factor）,whereas EPC was obtained in EPC medium（the presence of vascular endothelial growth factor）.SPC was identified as adherent cells positive for α-smooth muscle actin by indirect immunofluorescent staining.EPC was characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining.After 8 days cultured,SPC and EPC were treated with simvastatin（0.01-10 μmol/L） and then continued to be cultured for another 24 h.The migration of SPC and EPC were assayed with modified Boyden chamber assay.Results Simvastatin potently inhibited SPC migration.At concentration as low as 0.01 μmol/L,simvastatin significantly reduced SPC migration compared with control group（0.01 μmol/L simvastatin group versus control group： 39±3 vs.44±3,n=5,P0.05）.Being contary to SPC,incubation with simvastatin dose dependently increased EPC migration,at a maximum dose of 1.0 μmol/L（1.0 μmol/L simvastatin group versus control group：37±5 vs.6±3,n=5,P0.01）.Conclusions Simvastatin of equal dosage displays a differential effect on migration of SPC or EPC,inhibiting SPC migration but promoting EPC migration.