【作者】 屈顺林；

【导师】 杨向东； 黎健；

【作者基本信息】 南华大学 ， 病理生理学， 2005， 硕士

【摘要】 活性氧(reactive oxygen species,ROS)是调节血管功能状态的重要信号分子。业已证明,ROS可通过引起血管内皮细胞氧化损伤而在动脉粥样硬化发生过程中起重要作用。虽然黄嘌呤氧化酶、NADPH氧化酶(NOX)、线粒体电子传递链及一氧化氮合酶等均可产生ROS,但人血管系统中产生的ROS主要来源于NOX。最早在吞噬细胞中发现的NOX为gp91phox,近年来,在不同组织中如结肠、肾脏、脾脏及血管平滑肌细胞中,发现了新的gp91phox(现亦称为NOX2)同工酶,分别为NOX1、NOX3、NOX4和NOX5。最新研究提示NOX4可能作为血管内皮细胞中主要的NOX参与内皮细胞内ROS的生成。本研究首先用RT-PCR检测人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)、内皮细胞株(ECV304和HU-LT)和人脐动脉平滑肌细胞(human umbilical artery smooth muscle cells,HUASMCs)中NOX基因的表达,结果证实NOX4是人内皮细胞和平滑肌细胞中表达的主要NOX。在此基础上,观察HUVECs在高葡萄糖、高浓度低密度脂蛋白(low-density lipoprotein,LDL)、无血清和高血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)等培养条件下NOX4 mRNA、ROS水平和细胞凋亡的变化,分析NOX4表达与ROS生成之间的关系,进一步明确NOX4是否通过产生ROS参与内皮细胞损伤。 第一部分 高葡萄糖等对人内皮细胞NOX4表达、ROS水平、和凋亡的影响 目的 分析人脐静脉内皮细胞和人脐动脉平滑肌细胞中的NOX表达;研究在高葡萄糖、高LDL、无血清和高AngⅡ刺激HUVECs时细胞内ROS、NOX4 mRNA水平和凋亡的变化。更多还原

【Abstract】 Reactive oxygen species (ROS) are important regulators of vascular function. It has been demonstrated that ROS play an important role in the generation and progression of atherosclerosis through oxidative damage of vascular endothelial cells. Potential sources of endothelial ROS generation that are implicated in disease progress include mitochondria, xanthine oxidase (XO), uncoupled NO synthase, cytochrome p-450 enzymes, and NADPH oxidase (NOX) . ROS of human vascular system mostly derive from NADPH oxidase. Gp91phox was firstly found in phagocyte cells, and the novel gp91phox homologues of NOX families such as N0X1, N0X3, N0X4, N0X5 have been found in different organs (colon, kidney, spleen) and vascular smooth muscle cells in recent years. Latest studies indicate that N0X4 gene may be important in the ROS generation in endothelial cells. Firstly, mRNA expression of NOX gene was detected in human umbilical vein endothelial cells (HUVECs), ECV304, HU-LT and human umbilical artery smooth muscle cells (HUASMCs) by reverse transcription PCR, it was found that N0X4 is the major NOX gene in these cells. To further clarify the role of N0X4 gene in the apoptosis and ROS generation of HUVECs, we observed the change of N0X4 mRNA and ROS level as well as apoptosis in HUVECs stimulated by high concentration of glucose, low density lipoprotein(LDL), angiotensin II(AngII) and serum-free medium.Part I Effects of high concentration of glucose, LDL, Ang II and serum-free medium on N0X4 mRNA expression , ROS generation andapoptosis of HUVECsObjectives: To investigate the mRNA expression of NOX gene in HUVECs, ECV304, HU-LT and HUASMCs, then to elucidate the change of N0X4 mRNA expression, intracellular ROS level ,and apoptosis in HUVECs stimulated by high concentration ofglucose, low density lipoprotein(LDL), angiotensin II (Ang II) and serum-free medium. Methods: Morphology of HUVECs and HUASMCs was observed by inverted microscope, expression of factor Vffl related antigen in HUVECs was investigated by immunohistochemistry and immunofluorescence, expression of a-actin in HUASMCs was measured by immunohistochemistry. PcDNA3.1-N0Xl plasmid, U937 cell, HepG2 cell and PcDNA3.1-NOX4 plasmid were used as the positive control of mRNA expression of N0X1, N0X2, N0X3 and N0X4 respectively. NOX expression of HUVECs, HUASMCs, ECV304 and HU-LT was measured by RT-PCR. mRNA expression of N0X4, intracellular ROS level, and apoptosis in HUVECs were detected by RT-PCR, flow cytometry, Hoechst33342 staining respectively. Results: The results showed that there was expression of factor VBI related antigen in HUVECs and a-actin in HUASMCs. N0X4 was highly expressed in HUVECs, HUASMCs and HU-LT, while only a weak band of N0X2 was detected in HUVECs. However, all members of NOX were absent in ECV304. mRNA expression of N0X4, intracellular ROS level, and apoptosis were all increased in HUVECs stimulation with high glucose, LDL, Ang II and serum-free medium. Conclusions: (1) N0X4 is the major NOX families in HUVECs and HUASMCs.(2) The mRNA expression of NOX4 increases significantly along with the up-regulation of apoptosis, intracellular ROS level in HUVECs stimulated by high glucose, LDL, Ang II and serum-free medium.Part II Effects of high level expression of N0X4on HUVECs apoptosisObjectives: To elucidate the change of intracellular ROS level and apoptosis in HUVECs after transfection with PcDNA3.1-NOX4 plasmid.Methods: N0X4 mRNA expression in HUVECs and ECV304 transfection with PcDNA3.1-NOX4 plasmid or PcDNA3.1-GFP plasmid was measured by RT-PCR. Intracellular ROS level and apoptosis were detected with flow cytometry. The apoptosis of HUVECs and ECV304 was observed by Hoechst staining and TUNEL staining. Results: N0X4 mRNA expression and ROS level were increased significantly in更多还原