【摘要】 目的观察同型半胱氨酸(Hcy)对培养的人脐静脉内皮细胞(HUVEC)一氧化氮合酶(eNOS)活力及其基因表达的动态影响。方法10、30、100、300μmol.L-1Hcy与HUVEC分别培养24、48、72 h后,用HPLC测定细胞内不对称二甲基精氨酸(ADMA)的含量,反转录聚合酶链反应(RT-PCR)检测细胞内eNOS mRNA的表达,并分别测定细胞二甲基精氨酸二甲基氨基水解酶(DDAH)、eNOS的活力和NO的含量。结果HUVEC经不同浓度Hcy分别处理24、48、72 h后,其细胞内ADMA聚积增多,DDAH活性和eNOS活力降低,NO生成减少,且呈时间和浓度依赖性。但只有100μmo.lL-1Hcy与HUVEC作用72 h时,才引起eNOS mRNA表达的减少。结论Hcy对内皮功能的损伤可能通过抑制DDAH活性,引起ADMA聚积,从而降低eNOS活力,导致NO生成减少。此外,eNOS mRNA表达的抑制也是Hcy诱导的内皮功能障碍的机制之一。更多还原

【Abstract】 OBJECTIVE To observe the effects of homocysteine(Hcy) at different concentrations on activity and mRNA expression of endothelial nitric oxide synthase(eNOS) in cultured human umbical vein endothelial cell(HUVEC).METHODS HUVEC were cultured with Hcy at different concentrations(0,10,30,100,300 μmol·L-1) for 24,48 and 72 hours respectively.The content of asymmetric dimethylarginine(ADMA) was measured by RP-HPLC.RT-PCR was performed to detect the expression of eNOS mRNA.The activity of dimethylarginine dimethylaminohydrolase(DDAH),eNOS and the production of NO were analyzed at the same time.RESULTS After HUVEC were exposed to different concentrations of Hcy for 24,48 and 72 hours,ADMA concentration in the cell culture medium increased,mean while the activity of DDAH decreased in a dose-time-dependent fashion.The eNOS activity was inhibited,and the content of NO in the cell culture medium was significantly reduced compared with the control group(P < 0.05),but only when HUVEC was treated to 100 μmol·L-1 Hcy,did the expression of eNOS mRNA decrease.The expression of eNOS mRNA decreased.CONCLUSION The mechanism of homocysteine-induced endothelial dysfunction may exert on inhibiting DDAH expression,causing ADMA to accumulate,and lead to the decrease in eNOS activity and NO output.Its additional mechanism is the inhibition of the expression of eNOS mRNA.更多还原