【摘要】 目的研究不同时长下200μmol/L过氧化氢（H₂O₂）刺激对人脐静脉内皮细胞（human umbil-ical vein endothelial cell,HUVEC）衰老的影响,为氧化应激诱导细胞衰老模型提供参考。方法把HUVEC分为空白组和H₂O₂暴露组,暴露组分别在200μmol/L终浓度的H₂O₂作用0.5、1、3、6、12、24、48和96 h。噻唑蓝比色法（MTT）检测各组细胞活力;细胞衰老-β半糖苷酶染色（SA-β糖苷酶染色）检测细胞衰老情况;酶联吸附反应（enzyme linked immunoabsorbent assay,ELISA）测定各组细胞内皮素-1（endothelin-1,ET-1）、一氧化氮（nitric oxide,NO）含量;比色法测定各组细胞丙二醛（malondialdehyde,MDA）、超氧化物歧化酶（super oxidase dismutase,SOD）和谷胱甘肽过氧化物酶（glutathionperoxidase,GSH-px）水平;Western Blot检测Bax、Bcl-2、p21蛋白的表达情况。结果与空白组相比,H₂O₂暴露组短时间内（3 h以内）细胞增殖活性增高,少量细胞SA-β糖苷酶染色阳性,ET-1和NO分泌增加;随着刺激时间延长（3～24 h）,细胞增殖活性下降,SA-β糖苷酶染色阳性细胞增多,ET-1分泌增多,NO分泌减少（P <0.05）,p21表达逐渐增多,Bax、Bcl-2表达无明显差异;48～96 h细胞活力持续下降,SA-β糖苷酶染色阳性率无明显变化,ET-1、NO分泌下降,Bax表达逐渐增加,Bcl-2逐渐减少（P<0.05）,但p21表达无明显变化;与空白组相比,H₂O₂刺激后各组细胞MDA持续上升,SOD、GSH-px持续下降（P<0.05）。结论 200μmol/L的H₂O₂刺激24 h可导致HUVEC明显衰老,但随着刺激时间的延长,细胞凋亡增多。更多还原

【Abstract】 Objective To study the effect of different stimulated duration of 200μmol/L H₂O₂ on the senescence of human umbilical vein endothelial cells（HUVEC）,soas to provide reference for oxidative stressinduced senescence cell model.Method s The HUVEC were divided into control group and stimulated group which was subdivided into 8 groups according to the different stimulated duration of 200μmol/L H₂O₂ including0.5,1,3,6,12,24,48,96 h.MTT assays were performed to assess cell proliferation in each group;senescence-associatedβ-galactosidase（SA-β）staining was performed to measure the senescent cells;ELISA was used to measure the content of endothelin-1（ET-1）and nitric oxide（NO）;colorimetry assays were performed to assess the content of malondialdehyde（MDA）,super oxidase dismutase（SOD）and glutathionperoxidase（GSH-px）and western blot was performed to measure the expressions of Bax,Bcl-2 and p21.Results Compared with that in the control group,cell proliferation in H₂O₂ stimulated groups with a short-duration（<3 h）was significantly increased accompanied with very low positive rate of SA-βstaining and increased secretion of ET-1 and NO.With the prolonging of the stimulated duration of H₂O₂（3～24 h）,the cell proliferation decreased but the positive rate of SA-βstaining increased;the secretion of ET-1 increased but NO decreased,accompanied with increased expression of p21 but unchanged Bax and Bcl-2.In the 48～96 h,both the cell proliferation and the secretion of ET-1 and NO kept decreasing while there was no change of SA-βstaining.However,the expression of Bax increased but the Bcl-2decreased while there was no change of the p21.What′s more,compared with that in the control,MDA kept increasing but SOD and GSH-px decreasing in the stimulated groups.Conclusion The HUVEC stimulated with 200μmol/L H₂O₂ for 24 h may lead to significant senescence while over 24 h stimulation may lead to increased apoptosis.更多还原