Akt/eNOS信号途径调节内皮祖细胞存活和功能的实验研究

目的研究Akt/eNOS信号途径是否调节内皮祖细胞（EPC）的存活和功能。方法分离、培养EPC,然后与不同浓度氧化型低密度脂蛋白（oxLDL）、硝基精氨酸甲酯（L—NAME）或triciribine孵育48h,一部分EPC与左旋精氨酸预处理后,再与oxLDL孵育。然后,检测EPC凋亡率及迁移、黏附和管状结构形成能力,同时检测磷酸化Akt的蛋白表达、内皮型一氧化氮合酶（eNOS）的蛋白及mRNA表达、一氧化氮的产生。结果oxLDL剂量依赖性地诱导EPC凋亡,抑制EPC迁移、黏附及管状结构形成能力,L—NAME和triciribine具有与oxLDL相似的作用。oxLDL的作用能被左旋精氨酸抑制。oxLDL降低磷酸化Akt及eNOS蛋白表达,oxLDL剂量为50μg／ml时下降率分别为（664±4）％和（684±9）％。而且,oxLDL降低EPCeNOSmRNA的表达及一氧化氮的产生,oxLDL剂量为50μg／ml时eNOSmRNA表达的下降率为（594±14）％。结论oxLDL通过调节Akt／eNOS信号途径调节EPC的存活和功能。

oxLDL reduced endothelial progenitor cells survival and function via regulating Akt/eNOS signal pathway

OBJECTIVE: To investigate the association between Akt/eNOS signal pathway changes and the survival/function of endothelial progenitor cells (EPC) in the presence of oxLDL, L-NAME or triciribine. METHODS: After 14 d culture, EPC was stimulated with different concentrations of oxLDL, L-NAME or triciribine for 48 h. In one group, EPC was preincubated with L-arginine for 24 h and then exposed to 50 microg/ml oxLDL for 48 h. The survival and the ability of adhesion, migration and tube structure formation of EPC were observed and the level of phosphorylated Akt protein expression, eNOS protein and mRNA expression were assayed. RESULTS: Incubation with oxLDL at concentration 25 microg/ml or higher resulted EPC apoptosis, significantly reduced migratory rate, reduced adhesion to fibronectin and impaired ability of EPC to form tube structure in a dose-dependent manner. A simultaneous dose-dependent NO generation and Akt phosphorylation decrease as well as eNOS expression reduction at protein and mRNA levels were also observed. Pretreatment of EPC with L-arginine could attenuate these changes. CONCLUSION: oxLDL reduced EPC survival and function via regulating Akt/eNOS signal pathway.