CAJ | 학술논문

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（endothelial outgrowth cell,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、10 nmol/L rapamycin组（rapamycin组）、10μmol/L ryanodine预处理1h再加10 nmol/L的rapamycin组（ryanodine+ rapamycin组）、10 μmol/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( Western blot)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(Thr495)磷酸化水平。
목적 탐토리아낙감（ryanodine）대뢰파매소（rapamycin）유도적내피생장훈세포（endothelial outgrowth cell,EOC）공능급기상관총내피형일양화담합매(eNOS)단백표체급eNOS( Thr495)적린산화수평。방법 밀도제도리심법분리제혈단개핵세포，배양병확증EOC，면역조화법、형광염색법감정기내피세포특성。실험분위대조조、10 nmol/L rapamycin조（rapamycin조）、10μmol/L ryanodine예처리1h재가10 nmol/L적rapamycin조（ryanodine+ rapamycin조）、10 μmol/Lryanodine조（ryanodine조），각처리조여EOC작용24h，CCK8검측세포증식능력，Transwell소실검측세포천이능력。용특이성적총eNOS항체급린산화적eNOS( Thr495)[p-eNOS（Thr495）]항체적단백면역인적법( Western blot)검측총eNOS단백표체급eNOS( Thr495)린산화수평。결과 rapamycin조EOC적증식급천이균저우대조조（P균＜0.05），eNOS( Thr495)린산화수평고우대조조(P＜0.05)，총eNOS단백표체여대조조비교차이무통계학의의。Ryanodine+ rapamycin조EOC적증식급천이균고우rapamycin조（P균＜0.05），eNOS( Thr495)린산화수평저우rapamycin조(P＜0.05)，총eNOS단백표체여대조조비교차이무통계학의의。Ryanodine조EOC적증식、천이급총eNOS급eNOS( Thr495)린산화수평여대조조비교차이균무통계학의의。결론 rapamycin가억제EOC적증식급천이，증강eNOS(Thr495)린산화수평。Ryanodine가이개선rapamycin유도적EOC증식급천이능력적억제，강저eNOS(Thr495)린산화수평。
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 tnd expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine ( 10 μmol/L) for 1 h, and then treated with or without rapamycin ( 10nmoL/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 phosphoeNOS(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 downregnlated 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 ).ConclusionsRapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could bepartly reversed by cotreatment with ryanodine.