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目的观察外源性氧自由基(OFR)衍生的羟自由基(OH-)诱导培养的VECs凋亡及卡托普利(Captopril,Cap)的干预作用,探讨血管内皮细胞(VECs)凋亡(Apoptosis)与心血管疾病的关系。方法(1)采用改良的Jaffe法,进行人脐静脉血管内皮细胞(HUVECs)原代、传代培养;(2)取4～6代HUVECs,无血清条件下分别和10-5、10-4、10-3、10-2、10-1mol/L羟自由基(OH-)共孵育,观察细胞存活率、贴壁率、形态学、DNA凝胶电泳、DNA片段率及培养液一氧化氮(NO)含量变化;(3)观察10-3mol/LOH-诱导HUVECs凋亡及10-7、10-6、10-5mol/LCap干预后上述参数的变化。结果(1)正常培养HUVECs存活率>95%,贴壁率>90%;(2)外源性OH-明显降HUVECs存活率、贴壁率,且呈剂量依赖性;10-5、10-4、10-3mol/LOH-诱导HUVECs凋亡,DNA“梯形”以10-3mol/LOH-作用8～24h显著,DNA片段率、凋亡细胞百分率明显升高;而10-2、10-1mol/LOH-可诱导HUVECs坏死;(3)10-7、10-6、10-5mol/LCap干预明显提高OH-处理的HUVECs存活率、贴壁率,降低其凋亡细胞百分率、DNA片段率,使DNA“梯形”渐消失,NO分泌减少渐逆转。结论(1)采用改良的Jaffe法,可以建立HUVECs体外模型;(2)外源性OH-在较低浓度时可诱导培养HUVECs的典型凋亡;(3)Cap能减轻或抑制OH-诱导培养HUVECs的凋亡,恢复其NO分泌。
Objective To observe the apoptosis of VECs induced by exogenous oxygen free radical (OFR) -induced hydroxyl radical (OH-) and the effect of captopril (Cap) on the apoptosis of vascular endothelial cells (VECs) Relationship between Apoptosis and Cardiovascular Diseases. Methods (1) Primary and subcultured human umbilical vein endothelial cells (HUVECs) were cultured by modified Jaffe method. (2) HUVECs from 4 to 6 generations were harvested and cultured in serum-free medium with 10-5, 10-4, 10-3, 10-2, 10-1mol / L hydroxyl radical (OH-) co-incubated cells were observed survival rate, adherent rate, morphology, DNA gel electrophoresis, DNA fragment rate and the culture medium of nitric oxide NO). (3) The apoptosis of HUVECs induced by 10-3mol / LOH and the changes of these parameters after 10-7,10-6,10-5mol / L LCAP intervention were observed. Results (1) The survival rate of HUVECs in normal culture was> 95% and the rate of adherent was> 90%. (2) The exogenous OH- significantly reduced the survival rate and attachment rate of HUVECs in a dose- 4,10-3mol / LOH-induced apoptosis of HUVECs, DNA “trapezoid” to 10-3mol / LOH-effect of 8 ~ 24h significant DNA fragment rate, the percentage of apoptotic cells increased significantly; and 10-2,10-1 mol / LOH- can induce HUVECs necrosis; (3) 10-7,10-6,10-5mol / LCap intervention significantly increased the survival rate of OH-treated HUVECs, adherent rate, decreased the percentage of apoptotic cells, DNA fragment rate, The DNA “trapezoid” gradually disappear, NO secretion decreased gradually reversed. Conclusions (1) The modified Jaffe method can establish HUVECs in vitro model; (2) exogenous OH- can induce typical apoptosis of HUVECs cultured in low concentration; (3) Cap can reduce or inhibit OH-induced culture HUVECs apoptosis, restore its secretion of NO.