氧自由基对培养人脐静脉内皮细胞代谢产物的影响

Effects of oxygen-derived free radicals on metabolite of cultured human vascular endothelial cells derived from umbilical vein

目的:观察氧自由基对培养的人脐静脉内皮细胞产生的裂解不对称二甲精氨酸的酶--二甲精氨酸-二甲赖氨酸水解酶活性及表达的影响,以探讨不对称二甲精氨酸代谢机制及卡托普利的作用。方法:实验于2003-05/2004-06在南昌大学医学分子中心进行。采用改良的Jaffe法培养人脐静脉内皮细胞,取生长良好的3～6代人脐静脉内皮细胞分为4组:①空白对照组:加DMEM培养液。②氧自由基0.01,0.1mmol/L组:分别加入氧自由基0.01,0.1mmol/L。③卡托普利组:同时加入氧自由基0.1mmol/L及卡托普利(100mg/L)共孵。孵育24h后,检测上清液中一氧化氮、一氧化氮合酶活性、内皮细胞的代谢产物不对称二甲精氨酸浓度以及反应二甲精氨酸-二甲赖氨酸水解酶酶活性的L-瓜氨酸浓度,采用Westernblotting测定细胞裂解液中二甲精氨酸-二甲赖氨酸水解酶的蛋白表达。结果:①二甲精氨酸浓度:氧自由基0.01,0.1mmol/L组均高于空白对照组[(2.71±0.35),(4.78±0.67),(0.81±0.12)μmol/L,P<0.05,0.01],卡托普利组低于氧自由基0.1mmol/L组[(2.03±0.35)μmol/L,P<0.01]。②L-瓜氨酸浓度:氧自由基0.01,0.1mmol/L组均低于空白对照组(P<0.05,0.01),卡托普利组高于氧自由基0.1mmol/L组(P<0.01)。③一氧化氮浓度及一氧化氮合酶活性:氧自由基0.01,0.1mmol/L组均低于空白对照组(P<0.05,0.01),卡托普利组高于氧自由基0.1mmol/L组(P<0.01)。④二甲精氨酸-二甲赖氨酸水解酶的蛋白表达:各组间无差异(P>0.05)。结论:氧自由基培养下,内皮损伤,不对称二甲精氨酸的增加与二甲精氨酸-二甲赖氨酸水解酶的活性减弱有关,与其蛋白的表达无关。而卡托普利则通过增加二甲精氨酸-二甲赖氨酸水解酶活性促进不对称二甲精氨酸代谢,使一氧化氮合酶活性增加,抑制氧自由基对内皮功能的损伤。

AIM： To observe the effects of oxygen-derived free radical （OFR） activity and expression of dimethylarginine dimethylaminohydrolase （DDAH） of human vascular endothelial cell derived from umbilical vein, and to investigate the metabolic mechanism of asymmetriedimeth ylarginine （ADMA） as well as effect of captopril. METHODS： The study was performed in Medical Molecular Center of Nanchang University from May 2003 to June 2004. The human vascular endothelial cells of 3^rd-6^th passage, cultured with modified Jaffes＇ method, were rolled into the experiment and divided into 4 groups： ①Blank control group： Cells cultured with equivalence DMEM medium as control, ②0.01, 0.1 mmol/L OFR groups： Added with 0.01 mmol/L or 0.1 mmol/L OFR, respectively, and ③Captopril group： 0.1 mmol/l OFR groups were added with 100 mg/L captopril, together. Activities of nitrogen monoxide and nitricoxide synthase （NOS） and concentration of ADMA and L-citrulline that reflected the activity of DDAH in supernatant were detected after 24 hours exposure. DDAH expression in cell lysis buffer was measured with Western blotting. RESULTS： ①Concentration of ADMA： It was higher in the 0.01, 0.1 mmol/L OFR groups than that in the blank control group [（2.71±0.35）, （4.78 ±0.67）, （0.81 ±0.12）μmol/L,P 〈 0.05,0.01]. It was lower in the eaptopril group than that in the 0.1 mmol/L OFR group [（2.03±0.35 ）μmol/L, P 〈 0.01]. ②Coneentration of L-eitrulline： It was lower in the 0.01,0.1 mmol/L OFR groups than that in the blank control group （P 〈 0.05,0.01）. It was higher in the captopril group than that in the 0.1 mmol/L OFR group （P 〈 0.01）.③Coneentration of nitrogen monoxide and activity of NOS： It was lower in the 0.01,0.1 mmol/L OFR groups than that in the blank control group （P 〈 0.05,0.01）. It was higher in the eaptopril group than that in the 0.1 mmol/L OFR group （P 〈 0.01 ）. ④Expression of DDAH： There was no difference among groups （P 〉 0.05）. CONCLUSION： Endothelial dysfunction induced by OFR is associated with the increase of ADMA and reduction of DDAH activity, but not DDAH expression. Captopril can promote the degradation of ADMA through increasing activity of DDAH to inhibit the impairment of OFR on endothefia function.