课题背景：本课题得到了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）和湖南省医药卫生科研基金（B2006-101）的资助。课题开展了血管内皮细胞损伤过程中NADPH氧化酶基因表达图谱的分析，分析活性氧的产生及其对内皮细胞损伤的影响。

应用要点：作者最新的实验结果证实了在高浓度低密度脂蛋白等一些动脉粥样硬化危险因素的刺激下，作为主要NADPH氧化酶的NADPH氧化酶4通过产生高水平活性氧损伤血管内皮细胞，启动动脉粥样硬化的发生。通过对NADPH氧化酶4在内皮细胞氧化损伤过程中的作用机制观察，将为探寻保护内皮细胞免受氧化损伤的基因与因子提供新线索。

相关链接：①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。但活性氧产生的分子基础及氧化损伤内皮细胞的作用机制尚需进一步研究。②NADPH氧化酶的多亚基家族似乎对于内皮过氧化起着主导作用。相关链接：①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。但活性氧产生的分子基础及氧化损伤内皮细胞的作用机制尚需进一步研究。②NADPH氧化酶的多亚基家族似乎对于内皮过氧化起着主导作用。

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 24 h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；0.8 g/L 低密度脂蛋白组；1.6 g /L 低密度脂蛋白组；2.4 g/L低密度脂蛋白组。再以1.6 g/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，12 h处理组，24 h处理组，48 h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，Hoechst 33342染色分析细胞凋亡。
结果：①1.6 g/L低密度脂蛋白处理24 h后人脐静脉内皮细胞内活性氧生成量高于对照组（P < 0.05）。②1.6 g/L低密度脂蛋白处理24 h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（0.8，1.6，2.4 g/L）低密度脂蛋白处理人脐静脉内皮细胞 24 h和以1.6 g/L低密度脂蛋白处理人脐静脉内皮细胞不同时间（12，24和48 h）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，并促进细胞内活性氧生成和细胞凋亡。
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

屈顺林，丁莉，国汉邦，黄秀清，满永，王抒，杨向东，黎健．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 [J].中国组织工程研究与临床康复，2008，12(2):236-240
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-236(ps).pdf]

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

Abstract

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. ①Materials: 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. ②Procedure: 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. ③Assessments: 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: ①Compared with control group, ROS generation was increased in HUVECs after treatment with 1.6 g/L LDL for 24 hours (P < 0.05). ②The apoptosis in HUVECs treated with 1.6 g/L LDL for 24 hours was significantly higher than that in control group (P < 0.05). ③The RT-PCR results indicated that the mRNA expression of NOX4 was increased in HUVECs treated with different concentrations of LDL for different hours.
CONCLUSION: LDL could induce apoptosis through up-regulating NOX4 expression and intracellular ROS production in HUVECs.

Qu SL, Ding L, Guo HB, Huang XQ, Man Y, Wang S, Yang XD, Li J.Effects of high concentration of low-density lipoprotein on NADPH oxidase 4 mRNA expression, reactive oxygen species generation and apoptosis in vascular endothelial cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):236-240(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-236(ps).pdf]