理阿诺碱增强雷帕霉素诱导的内皮生长晕细胞功能并降低内皮型一氧化氮合酶（Thr495）的磷酸化水平

Ryanodine downregulates the expression of p-eNOS （Thr495） and improves the functions of rapamycin treated endothelial outgrowth cells

目的探讨理阿诺碱（ryanodine）对雷帕霉素（rapamycin）诱导的内皮生长晕细胞（endothelialoutgrowthcell，EOC）功能及其相关总内皮型一氧化氮合酶（eNOS）蛋白表达及eNOS（Thr495）的磷酸化水平。方法密度梯度离心法分离脐血单个核细胞，培养并扩增EOC，免疫组化法、荧光染色法鉴定其内皮细胞特性。实验分为对照组、10nmol/L rapamycin组（rapamycin组）、10μmoL／Lryanodine预处理1h再加10nmol/L的rapamycin组（ryanodine＋rapamycin组）、10μmoL／Lryanodine组（ryanodine组），各处理组与EOC作用24h，CCK8检测细胞增殖能力，Transwell小室检测细胞迁移能力。用特异性的总eNOS抗体及磷酸化的eNOS（Thr495）[p-eNOS（Thr495）]抗体的蛋白免疫印迹法（Westernblot）检测总eNOS蛋白表达及eNOS（Thr495）磷酸化水平。结果rapamycin组EOC的增殖及迁移均低于对照组（P均〈0．05），eNOS（Thr495）磷酸化水平高于对照组（P〈0．05），总eNOS蛋白表达与对照组比较差异无统计学意义。ryanodine＋rapamycin组EOC的增殖及迁移均高于rapamycin组（P均〈0．05），eNOS（Thr495）磷酸化水平低于rapamycin组（P〈0．05），总eNOS蛋白表达与对照组比较差异无统计学意义。ryanodine组EOC的增殖、迁移及总eNOS及eNOS（Thr495）磷酸化水平与对照组比较差异均无统计学意义。结论rapamycin可抑制EOC的增殖及迁移，增强eNOS（Thr495）磷酸化水平。ryanodine可以改善rapamycin诱导的EOC增殖及迁移能力的抑制，降低eNOS（Th1495）磷酸化水平。

Objective To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells （EOCs）. Methods The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine （10 iJunol/L） for 1 h, and then treated with or without rapamycin （10 nmol/L） for 24 h. Proliforation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho- eNOS（Thr495） antibody. Results Compared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS （Thr495） protein was significantly upregulated in rapamycin group （ P 〈 0. 05 ）, expression of total eNOS was not affected by rapamycin （ P 〉 0.05）. Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS（Thr495） protein was significantly downregulated in ryanodine ＋ rapamycin group（P 〈0. 05）. The proliferation and migration capacities, the phosphorylation of eNOS（Thr495 ） protein and the expression of total eNOS were not affected by ryanodine alone （P 〉 0. 05 ）. Conclusions Rapamyein reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS （Thrg95） protein of EOCs and these effects could bepartly reversed by cotreatment with ryanodine.