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目的探讨重组血管紧张素转换酶2(ACE2)基因转染对内皮源性一氧化氮(NO)合酶(eNOS)磷酸化水平的影响。方法克隆和构建含人ACE2基因全长的重组质粒(pACE2),并将之转染入人血管内皮细胞中;采用实时定量PCR和Western印迹技术检测转染细胞中的ACE2、eNOS的表达情况,同时使用硝酸还原酶比色法测定细胞中NO含量。结果血管紧张素II(AngII,100nmol/L)加入细胞后可抑制由胰岛素刺激的Ser1177-eNOS磷酸化与NO生成(n=5～6,P<0.01)。pACE2基因转染可逆转细胞中由AngII对胰岛素诱导eNOS磷酸化的抑制效应,同时伴NO水平上升(n=5～6,P<0.01)。结论ACE2过表达可明显改善人血管内皮细胞中eNOS磷酸化的表达,伴有NO生成增加,提示ACE2可能通过调节血管AngII/NO的平衡,在改善血管内皮功能和胰岛素抵抗中起重要功效。
Objective To investigate the effect of recombinant adenovirus 2 (ACE2) gene transfection on phosphorylation of endothelial nitric oxide synthase (eNOS). Methods The recombinant plasmid containing human ACE2 gene (pACE2) was cloned and constructed and transfected into human vascular endothelial cells. The expression of ACE2 and eNOS in transfected cells was detected by real-time PCR and Western blotting, At the same time nitrate reductase colorimetric determination of NO content in cells. Results AngII (100 nmol / L) could inhibit the phosphorylation of Ser1177-eNOS stimulated by insulin and NO production (n = 5-6, P <0.01). Transfection of pACE2 gene could reverse the inhibitory effect of AngII on insulin-induced eNOS phosphorylation in cells, accompanied by an increase of NO level (n = 5-6, P <0.01). Conclusion Overexpression of ACE2 can significantly improve the phosphorylation of eNOS in human vascular endothelial cells with increased NO production, suggesting that ACE2 may play an important role in improving endothelial function and insulin resistance by regulating the balance of AngII / NO.