ADMA对内皮生长晕细胞凋亡及其相关p38丝裂素活化蛋白激酶表达的影响

Effects of asymmetric dimethylarginine on apoptosis and expression of p38 mitogen-activated protein kinase in endothelial outgrowth cells

目的:探讨非对称性二甲基精氨酸(ADMA)对内皮生长晕细胞(EOCs)凋亡和黏附能力的影响以及凋亡相关p38丝裂素活化蛋白激酶(p38MAPK)表达水平的变化。方法:分离脐血中单个核细胞并原代培养扩增内皮生长晕细胞,免疫细胞化学染色和荧光染色法鉴定其内皮细胞特性。将浓度为0、1、5、10、30μmol/L ADMA与内皮生长晕细胞作用48 h,流式细胞仪检测细胞凋亡率,DAPI染色及AnnexinV/PI双染观察凋亡细胞核形态变化。在倒置显微镜下观察计数重黏附细胞数来测定细胞黏附能力。用特异性的phospho-p38MAPK抗体的W esternb lotting检测p38MAPK的活性。结果:采用贴壁法培养的细胞具有多种内皮细胞特性。ADMA(1-30μmol/L)诱导内皮生长晕细胞的凋亡(P<0.01),同时ADMA(5-30μmol/L)使phospho-p38MAPK蛋白表达增加,两者作用均呈浓度依赖性。ADMA作用组和对照组相比,激光共聚焦显微镜下可见更显著的细胞凋亡形态学改变。除1μmol/L ADMA外,5、10、30μmol/L ADMA均可抑制内皮生长晕细胞的黏附能力。结论:ADMA能诱导内皮生长晕细胞发生凋亡并抑制其黏附能力,ADMA诱导内皮生长晕细胞发生凋亡可能与p38 MAPK磷酸化水平上升有关。

AIM： To investigate the effects of asymmetric dimethylarginine （ADMA） on apoptosis, adhesion ability and phosphorylation of p38 mitogen-activated protein kinase in endothelial outgrowth cells （EOCs）. METHODS： The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, and induced into EOCs and then expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were treated with different concentrations of ADMA （0, 1, 5, 10, 30 μmol/L） for48 h. Apop- totic incidences of EOCs were quantitatively determined by flow cytometry. The morphologic changes of the apoptotic cells were visualized by DAPI staining and Annexin - V/PI staining. Adhesion ability of EOCs was measured by replacing the cells on dishes and adherent cells were counted under the inverted microscopy. The p38MAPK activity was evaluated by im- munoblotting technique with phospho -p38MAPK antibody. RESULTS： EOCs possessed many endothelial characteristics. Immunostaining showed that the surface antigen factor VIII, CD34 and Flk - 1 were positive. The fluorescent staining revealed that EOCs were double positive for Dil - ac - LDL - uptaking and FITC - UEA - I - lectin binding. ADMA （ 1 - 30 μmol/L） induced apoptosis of EOCs in a dose - dependent manner （ P 〈 0. 01 ）. Obvious change of apoptotic morphology in EOCs incubated with ADMA was observed with DAPI staining and Annexin- V/PI staining. ADMA （5 -30 μmol/L） inhibited the adhesion ability of EOCs, whereas ADMA at concentration of 1 taxnol/L had no effect. ADMA （5 -30 p, mol/L） induced phosphorylation of p38MAPK in a dose - dependent manner （ P 〈 0.01 ）. CONCLUSION： ADMA induces apoptosis and inhibits adhesion ability in EOCs. Activated p38MAPK might be involved in the course of apoptotic effects of ADMA.