高浓度低密度脂蛋白对血管内皮细胞NADPH氧化酶4 mRNA水平、活性氧产生及细胞凋亡的影响  

Effects of high concentration of low-density lipoprotein on NADPH oxidase 4 mRNA expression,reactive oxygen species generation and apoptosis in vascular endothelial cells

作　　者：屈顺林[1,2] 丁莉[1,2] 国汉邦[1] 黄秀清[1] 满永[1] 王抒[1] 杨向寿[2] 黎健[1] 

机构地区：[1]卫生部北京医院老年医学研究所,卫生部老年医学重点实验室,北京市100730 [2]南华大学医学院,湖南省衡阳市421001

出　　处：《中国组织工程研究与临床康复》2008年第2期236-240,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30440065、30572082);北京市自然科学基金资助项目(7052059);湖南省医药卫生科研基金(B2006-101)~~

摘　　要：目的:低密度脂蛋白进入血管内皮细胞后可刺激细胞产生活性氧,在内皮细胞的生长与损伤过程中起关键作用。实验拟进一步观察高浓度低密度脂蛋白对人脐静脉内皮细胞活性氧、NADPH氧化酶4 mRNA水平和凋亡的影响。方法:实验于2004-12/2006-02在卫生部老年医学重点实验室完成。①实验材料:脐带来自卫生部北京医院产科,产妇或家属签署知情同意书;低密度脂蛋白由卫生部北京医院老年医学研究所生化室经过超速离心提取。②实验过程及分组:分离人脐静脉内皮细胞。先以不同浓度的低密度脂蛋白处理人脐静脉内皮细胞24h以选取最佳的浓度,观察NADPH氧化酶4表达,分为:不加低密度脂蛋白的对照组;0.8g/L低密度脂蛋白组;1.6g/L低密度脂蛋白组;2.4g/L低密度脂蛋白组。再以1.6g/L低密度脂蛋白浓度处理人脐静脉内皮细胞不同的时间以选取最佳的处理时间观察NADPH氧化酶4表达,分为:不加低密度脂蛋白的对照组,12h处理组,24h处理组,48h处理组。以最佳浓度和最佳时间处理人脐静脉内皮细胞,并观察其NADPH氧化酶4表达、活性氧生成和凋亡。③实验评估:反转录-聚合酶链反应检测人脐静脉内皮细胞中NADPH氧化酶的表达;流式细胞仪检测胞内活性氧生成量和细胞凋亡率,Hoechst33342染色分析细胞凋亡。结果:①1.6g/L低密度脂蛋白处理24h后人脐静脉内皮细胞内活性氧生成量高于对照组(P<0.05)。②1.6g/L低密度脂蛋白处理24h组细胞凋亡也比对照组明显增加(P<.05)。③用反转录-聚合酶链反应检测不同浓度(0.8,1.6,2.4g/L)低密度脂蛋白处理人脐静脉内皮细胞24h和以1.6g/L低密度脂蛋白处理人脐静脉内皮细胞不同时间(12,24和48h)时NADPH氧化酶4 mRNA水平变化,结果显示与不加低密度脂蛋白的对照组相比,各组NADPH氧化酶4 mRNA表达均增强。结论:低密度脂蛋白上调人脐静脉内皮细胞内NADPH氧化酶4 mRNA表达,AIM： Low-density lipoprotein （LDL） can induce the generation of reactive oxygen species （ROS） of vascular endothelial cells and has a principal role in the growth and injuries of endothelium. The aim of this study was to investigate the effect of high concentration of LDL on ROS generation, mRNA level of NADPH oxidase 4 （NOX4） and apoptosis in human umbilical vein endothelial cells （HUVECs）. METHODS： The experiment was carried out in the Key Laboratory of Geriatrics, Ministry of Health from December 2004 to February 2006. （1）Materials： Umbilical cords were collected from Maternity Department of Beijing Hospital with informed consents. LDL was isolated by sequential ultracentrifugation in Biochemistry Laboratory of Beijing Institute of Geriatrics, Beijing Hospital. （2） Procedure： The optimum concentration and time of LDL treatment were selected, and then isolated HUVECs were treated for 24 hours with different concentrations of LDL. The NOX4 expression was observed after selecting an optimal concentration. There were control group, 0.8 g/L LDL group, 1.6 g/L LDL group and 2.4 g/L LDL group. The NOX4 expression was observed after HUVECs treated with 1.6 g/L LDL for an optimal time. There were control group, 12-hour treatment group, 24-hour treatment group and 48-hour treatment group. ROS generation, NOX4 and apoptosis in HUVECs treated with optimized LDL concentration and proper hours were detected. （3）Assessments： The NOX4 expression was measured by reverse transcription-polymerase chain reaction （RT-PCR）. Intracellular ROS level and apoptotic rate were determined by Flow Cytometry （FCM）. Hoechst 33342 staining was used to analyze the apoptosis in HUVECs. RESULTS： （1）Compared with control group, ROS generation was increased in HUVECs after treatment with 1.6 g/L LDL for 24 hours （P 〈 0.05）. （2）The apoptosis in HUVECs treated with 1.6 g/L LDL for 24 hours was significantly higher than that in control group （P 〈 0.05）. （3）The RT-PCR results indi

关 键 词：内皮细胞 脐静脉 脂蛋白类 NADPH氧化酶 活性氧 细胞凋亡 组织构建 

分 类 号：R329.4[医药卫生—人体解剖和组织胚胎学]