不同时长的过氧化氢刺激对人脐静脉内皮细胞衰老的影响  

Effects of different stimulated duration of hydrogen peroxide on senescence of human umbilical vein endo⁃thelial cells

作　　者：林朴卿 阮云军[1] LIN Puqing;RUAN Yunjun(Department of Gerontology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学南方医院老年病科,广州510515

出　　处：《实用医学杂志》2021年第10期1263-1267,共5页The Journal of Practical Medicine

基　　金：广东省自然科学基金(编号:2016A030313755)。

摘　　要：目的研究不同时长下200μmol/L过氧化氢(H_(2)O_(2))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H_(2)O_(2)暴露组,暴露组分别在200μmol/L终浓度的H_(2)O_(2)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,H_(2)O_(2)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,H_(2)O_(2)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H_(2)O_(2)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。Objective To study the effect of different stimulated duration of 200μmol/L H_(2)O_(2)on the senescence of human umbilical vein endothelial cells(HUVEC),soas to provide reference for oxidative stress⁃induced senescence cell model.Methods 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 H_(2)O_(2)including 0.5,1,3,6,12,24,48,96 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 H_(2)O_(2)stimulated groups with a short⁃duration(<3 h)was significantly increased accom⁃panied with very low positive rate of SA⁃βstaining and increased secretion of ET⁃1 and NO.With the prolonging of the stimulated duration of H_(2)O_(2)(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⁃2 decreased 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 H_(2)O_(2)for 24 h may lead to significant senescence while over 24 h stimulation may lead to increased apoptosis.

关 键 词：不同时长 过氧化氢 人脐静脉内皮细胞 衰老 

分 类 号：R965[医药卫生—药理学]