【摘要】 目的:观察卡维地洛对氧自由基(OFR)培养的人脐静脉内皮细胞(HUVECs)二甲精氨酸-二甲赖氨酸水解酶(DDAH)活性及表达的影响,以探讨卡维地洛对不对称二甲精氨酸(ADMA)代谢机制的影响。方法:采用改良的Jaffe法培养原代人脐静脉内皮细胞(HUVECs),取生长良好的3-6代HUVECs用于实验,分为①空白对照组:加DMEM培养液;②OFR组:加入OFR(0.01 mmol/L,0.1 mmol/L);③OFR+卡维地洛组:同时加入0.1 mmol/L OFR及卡维地洛(10μmol/L)共孵24 h后,检测上清液中一氧化氮(NO)、内皮素(ET)、ADMA含量、L-胍氨酸(L-cit)浓度及一氧化氮合酶(NOS)活性。用Western blotting法测定细胞裂解液中二甲基精氨酸-二甲基赖氨酸水解酶(DDAH)的蛋白表达。结果:OFR条件培养下,内皮细胞的代谢产物ADMA、ET的量均高于空白对照组,而NO的量及NOS的活性少于空白对照组;反映DDAH酶活性的L-cit浓度显著降低,且有浓度依赖性,而DDAH的表达无明显变化。卡维地洛干预组的ADMA、ET的量均低于OFR组,NOS活性及NO、L-cit浓度明显高于OFR组。结论:OFR培养下,内皮损伤ADMA的增加与DDAH的活性减弱有关,而与DDAH的表达无关。卡维地洛通过增加DDAH活性促进ADMA代谢,使NOS活性增加,抑制OFR对内皮功能的损伤。更多还原

【Abstract】 AIM: To observe the effects of oxidized free radical(OFR) on dimethylarginine dimethylaminohydrolase(DDAH) activity and concentration in human umbilical vein endothelial cells(HUVECs),and to investigate the metabolic mechanism of endogenous NOS inhibitor and the role of carvedilol.METHODS: HUVECs of 3-6th passage,cultured with modified Jaffes’ method,were divided into three groups:(1) cells cultured with equivalent DMEM medium as control;(2) OFR intervention groups,0.01 mmol/L,or 0.1 mmol/L OFR was added respectively;(3) drug intervention groups: 0.1 mmol/L OFR plus 10 μmol/L carvedilol.ADMA,nitric oxide(NO),endothim(ET),L-citrulline concentrations and the activity of NOS in conditioned medium were measured after 24 h exposure.ADMA concentration in the conditioned medium was determined by high-performance liquid chromatography.Western blotting was performed to evaluate DDAH expression.RESULTS: Compared with control group,ADMA and ET concentrations were increased,while the level of NO and the activity of NOS decreased and relevant to the concentrations of OFR.We assayed DDAH activity by determining L-citrulline formation from ADMA.The concentration of L-citrulline was decreased,while the DDAH expression had no obvious change.With the role of carvedilol,ADMA,ET concentrations were decreased,while the level of NO,L-citrulline and the activity of NOS increased.CONCLUSION: Endothelial dysfunction induced by OFR is associated with the increase in ADMA concentration and reduction of DDAH activity,but not DDAH expression.Carvedilol promotes the degradation of ADMA through increasing activity of DDAH and improving endothelium function.更多还原