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目的观察阿司匹林对外周血内皮祖细胞(endothelial progenitor cells,EPCs)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7d后收集贴壁细胞,加入不同浓度阿司匹林(1、2、5、10 mmol/L)分别培养3、6、12、24 h。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,将其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形成试剂盒来观察EPCs的增殖能力、迁移能力、黏附能力和体外血管形成能力,免疫印迹杂交法(Western blot)半定量测定诱导型一氧化氮合酶(iNOS)含量。结果阿司匹林减少外周血EPCs数量,并且EPCs数量随阿司匹林浓度和作用时间增加而减少。其增殖能力、迁移能力、黏附能力、体外血管形成能力和iNOS含量亦随阿司匹林浓度和作用时间增加而降低。结论阿司匹林通过EPCs中iNOS含量,降低EPCs的增殖能力、迁移能力、黏附能力和体外血管形成能力,从而抑制内皮细胞的生成。
Objective To observe the effect of aspirin on endothelial progenitor cells (EPCs). Methods Mononuclear cells were obtained from peripheral blood by density gradient centrifugation and inoculated on human fibronectin-coated plates. After 7 days of culture, adherent cells were collected and treated with different concentrations of aspirin (1, 2, 5, 10 mmol / L) were cultured 3,6,12,24 h. Confocal laser scanning microscopy identified FITC-UEA-I and Dil-acLDL double-stained positive cells as differentiated EPCs, which were counted under an inverted fluorescence microscope. The proliferation, migration, adhesion and in vitro angiogenesis of EPCs were observed by MTT assay, modified Boyden chamber, adhesion assay and in vitro angiogenesis kit, respectively. Quantitative determination of inducible nitric oxide synthase (iNOS) content. Results Aspirin reduced the number of EPCs in peripheral blood and the number of EPCs decreased with the increase of aspirin concentration and duration of action. Its proliferation, migration, adhesion, in vitro vascular formation and iNOS content also decreased with aspirin concentration and duration of action. Conclusion Aspirin can inhibit the formation of endothelial cells through the iNOS content in EPCs and reduce the proliferation, migration, adhesion and in vitro angiogenesis of EPCs.