法舒地尔改善内皮祖细胞功能活性的机制研究

Promotion effects of fasudil on endothelial progenitor cell function

目的:研究法舒地尔对内皮祖细胞(endothelial progenitor cells,EPCs)功能活性及一氧化氮(NO)合成的影响。方法:利用密度梯度离心法分离、培养人外周血单个核细胞,由FITC-UEA-I和Dil-acLDL双染色鉴定正在分化的EPCs。将分离、培养的EPCs分为对照组、法舒地尔组(10μmol/L)和内皮型一氧化氮合酶抑制剂(L-NAME,100μmol/L)干预组。分别采用免疫荧光染色计数法、Transwell小室法、粘附实验以及体外小管形成实验观察法舒地尔干预后EPCs增殖、迁移、粘附以及血管形成能力的变化情况,同时采用Greiss法检测法舒地尔处理后EPCs合成NO的变化情况,应用Western blot计数检测法舒地尔处理EPCs后一氧化氮合酶(eNOS)和磷酸化eNOS(Ser1179)蛋白的表达变化情况。结果:与对照组相比,法舒地尔组EPCs增殖、迁移、粘附以及血管形成等功能活性显著提高,而在L-NAME组,法舒地尔改善EPCs功能活性的作用受到明显的抑制。另外,EPCs经法舒地尔处理后,NO的合成分泌水平以及磷酸化eNOS(Ser1179)表达水平均明显高于对照组(P<0.05)。结论:法舒地尔具有促进EPCs增殖、迁移、粘附等功能的作用,其主要机制可能与法舒地尔促进eNOS的活性,进而增加NO的合成分泌有关。

Objective： Abstract Objective： To investigate the effects of fasudil on endothelial progenitor cells （EPCs） functional activities. Method： Peripheral blood derived mononuclear cells containing EPCs were isolated from healthy volunteers and then cultured on fibronectin-coated dishes with EBM-2 medium. The cells were cul tured alone （control groups）, with fasudil （10 μmol/L）, and with fasudil plus endothelial NO synthase（eNOS）in- hibitor （N-nitro-L-argininemethyl ester, L NAME, 100 μmol/L）. The proliferative, migratory,adhesive and an- giogenic activities of EPCs in vitro were determined by immunofluorescence staining assay, transwell cell migration assay, adhesive assay and tube formation assay, respectively. The production of NO was assessed by Griess reac- tion. The expression of eNOS and phosphorylation eNOS（Ser1179） was determined by Western blot. Result：DiI- AcLDL uptake and lectin staining r6vealed that these proliferation colonies were EPCs. The proliferative, migra- tory, adhesive and angiogenic activities of EPCs were significantly enhanced after treated with fasudil, which could be significantly blocked by eNOS inhibitor（L-NAME）. Meanwhile, the concentration of NO and the expression of phosphorylation eNOS（Ser1179）were significantly upregulated by the treatment of fasudil. Conclusion： Fasudil could improve the functional activities of EPCs by promoting eNOS activity and enhancing nitric oxide secretion.