【作者】 于世勇；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 研究背景随着生活水平的提高、人口老龄化趋势的加快以及不良生活方式的影响,冠状动脉粥样硬化性心脏病(冠心病)在我国和全球均呈明显的增长趋势,是当前社会人群主要死因,且患病率和死亡率仍在逐年上升。因此明确冠心病的相关危险因素及致病机理对于冠心病的防治具有重要的意义。经过多年的研究,人们对冠心病的认识有了巨大的进步,确定了吸烟、高血压、血脂紊乱、糖尿病等传统危险因素,同时新的危险因素也不断被发现,如高尿酸、高同型半胱氨酸等。近年来,晚期糖基化终末产物(advanced glycation end products,AGEs)在冠心病发生发展中的作用逐渐引起世人的瞩目。AGEs促动脉粥样硬化形成的主要机制在于诱发细胞内活性氧(reactive oxygen species,ROS)的生成增多,进而导致内皮功能失调,并引起内皮细胞损伤更为特异性的改变即内皮细胞活化。因此抑制ROS生成将有效减轻内皮细胞氧化损伤进而对抗AGEs促动脉粥样硬化效应。但是在生理和病理情况下,体内有多种酶参与了ROS的生成,包括线粒体呼吸链酶复合体、黄嘌呤氧化酶、细胞色素P450酶、解耦联的NOS、吞噬细胞髓过氧化物酶系统以及NADPH氧化酶,目前研究表明NADPH氧化酶在多种因素刺激内皮细胞生成ROS的过程中起主要作用。那么,在AGEs诱发内皮细胞ROS生成增加过程中,是否通过NADPH氧化酶这一途径?这是本课题所要研究的目的之一。如果NADPH氧化酶在这一过程中具有特异性调节作用,抑制NADPH氧化酶将减轻AGEs对内皮细胞的氧化损伤。但是NADPH氧化酶体内分布广泛,尤其是吞噬细胞,在宿主抵御微生物的非特异性反应中发挥重要作用,抑制后会引起严重后果,如何特异性的抑制内皮细胞NADPH氧化酶?近来的研究表明NADPH氧化酶存在多种亚型,包括Nox1、Nox2、Nox3、Nox4、Nox5以及Duox1和Duox2,其组织分布具有高度特异性,Nox4是内皮细胞NADPH氧化酶的主要表现形式。Nox4在AGEs诱导内皮细胞氧化损伤过程中对ROS生成是否具有调控作用,目前尚不清楚。氧化应激在内皮细胞活化和功能失调的发生中具有重要作用,对远期心血管疾病的发病率和死亡率有深远的意义,但是临床上多种抗氧化剂预防心血管终点事件的临床试验均以失败告终,表明单纯清除自由基这一治疗途径是有缺陷的,而抑制特异的ROS生成酶可能更为恰当。NADPH氧化酶对内皮细胞ROS生成的高度特异性调节作用显示出其重要性,有必要进行深入的研究,为动脉粥样硬化的防治提供理论依据,也为选择新的药物靶点提供实验依据。研究目的1、建立人冠状动脉内皮细胞活检方法,从病变血管获得一定数量的有活性的冠状动脉内皮细胞,为准确检测冠状动脉内皮细胞在疾病状态下的病理变化提供标本来源。2、分析血清AGEs与冠状动脉粥样硬化病变程度及冠状动脉内皮细胞氧化损伤的相关性,探讨AGEs促冠状动脉粥样硬化过程中的可能机制。3、观察AGEs与血管内皮细胞氧化损伤的量效关系。4、比较各种氧化酶在AGEs诱导血管内皮细胞ROS生成中的作用大小,明确调控ROS生成的氧化酶类型。5、观察NADPH氧化酶亚型Nox4在AGEs诱导血管内皮细胞氧化损伤过程中的作用,探寻特异性抑制血管内皮细胞ROS生成的关键靶点。实验方法1、人冠状动脉内皮细胞活检方法的建立及鉴定:通过冠状动脉介入手术中的导丝获取冠状动脉内皮细胞,进而应用免疫磁珠进行分选,瑞氏染色后倒置相差显微镜下进行细胞形态学观察并计数,vWF抗体及CD31抗体免疫荧光法进行鉴定,通过3种方法评价内皮细胞的活性:(1)荧光素乙酰乙酸盐(Fluorescein diacetate,FDA)/碘化丙啶(Propidium Iodide,PI)双荧光染色法检测细胞膜的完整性;(2)DiI-acLDL摄取实验观察内皮细胞的代谢活性;(3)Annexin V-FITC/PI双荧光染色法检测细胞凋亡。并尝试对活检细胞进行体外培养。2、冠心病患者冠状动脉病变程度及冠状动脉内皮细胞氧化损伤与血清AGEs的关系:根据美国心脏病协会所规定的冠状动脉血管图像计分评价标准,采用Gensini积分系统,对冠心病患者冠状动脉病变程度进行评定;ELISA法测定血清AGEs含量;利用冠状动脉内皮细胞活检技术,获取冠状动脉内皮细胞,通过荧光染料2’,7’-二氯二氢荧光素二乙酯(2’,7’-dichlorodihydrofluorescein diacetate,DCFH-DA)测定细胞内ROS水平;采集冠状静脉窦血液标本,测定血清一氧化氮(nitric oxide,NO)含量,以此反映冠状动脉内皮细胞功能。分别分析冠状动脉病变程度、冠状动脉内皮细胞ROS水平、冠状静脉窦血清NO浓度与血清AGEs含量之间的相关性。3、AGEs对血管内皮细胞的氧化损伤效应:制备AGEs;I型胶原酶消化法分离培养脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),vWF抗体免疫荧光法进行鉴定;将HUVECs接种至24孔培养板,待细胞生长至融合状态后,无血清培养液培养24 h。分别加入100μg/mL、200μg/mL、300μg/mL、400μg/mL、500μg/mL、600μg/mL、700μg/mL、800μg/mL AGEs,在1 h、2h、4 h、8h、16 h及24 h ,通过DCFH-DA法检测细胞内ROS水平,流式细胞仪分析细胞表面细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)表达情况,并确定最佳的AGEs作用浓度和时间。4、比较各种氧化酶在AGEs诱导血管内皮细胞ROS生成中的作用大小:将HUVECs接种至24孔培养板,待细胞生长至融合状态后,无血清培养液培养24 h。分为AGEs刺激组:加入600μg/mL AGEs;AGEs+氧化酶阻断剂组:应用鱼藤酮(2μmol/L)抑制线粒体呼吸链酶复合体I,噻吩甲酰三氟丙酮(thenoyltrifluoroacetone,TTFA;10μmol/L)抑制线粒体呼吸链酶复合体II,抗霉素A(2μmol/L)抑制线粒体呼吸链酶复合体III,别嘌醇(100μmol/L)抑制黄嘌呤氧化酶,左旋硝基精氨酸甲酯(Nitro-L-arginine methyl ester,L-NAME;100μmol/L)抑制一氧化氮合酶(nitric oxide synthase,NOS),二亚苯基碘(diphenylene iodonium,DPI;10μmol/L)抑制NADPH氧化酶,以上氧化酶抑制剂在加入AGEs30分钟之前分别加入,并与AGEs一起与HUVECs共同孵育直至最后检测;以不加任何刺激剂的DMEM培养基组作为对照组。在16h时检测各组细胞内ROS水平。5、Nox4在AGEs致内皮细胞氧化损伤中的作用: RT-PCR法检测Nox4 mRNA表达,免疫荧光染色及Western blot法检测Nox4蛋白表达,观察AGEs刺激后Nox4的表达变化。并针对Nox4 mRNA,合成siRNA,应用Lipofectamine 2000转入内皮细胞,特异性阻断Nox4,测定血管内皮细胞ROS生成及细胞表面ICAM-1表达情况。结果1、人冠状动脉内皮细胞活检方法的建立及鉴定、体外培养:在37例行冠状动脉介入治疗的患者中,我们获得了37根冠脉导丝并进行了冠状动脉内皮细胞的磁珠分选。瑞氏染色后显微镜下观察活检所得内皮细胞呈圆形或卵圆形,直径大于20μm,胞浆呈粉红色,胞核呈紫红色,周围结合有较多磁珠颗粒。每张涂片可检测到3～14个细胞,平均9.6个,每个患者可通过导丝获取96个细胞左右。经vWF抗体及CD31抗体免疫荧光染色后,荧光显微镜下可见细胞浆内有vWF的表达,同时细胞膜有CD31的表达,表明活检所得细胞为内皮细胞。FDA/PI双染后荧光显微镜下观察胞膜完整的细胞呈现明亮的绿色,而胞膜破坏的细胞表现为红色的细胞核。具有代谢活性的细胞表现出摄取DiI-acLDL的能力,荧光显微镜下胞浆内可见红色颗粒状物质。所获取的冠状动脉内皮细胞中,96%表现出细胞膜的完整性和代谢活性。经Annexin V-FITC/PI染色后,约7%表现为早期凋亡细胞(Annexin V~+/PI~-),约2%表现为晚期凋亡细胞或坏死细胞(Annexin V~+/PI~+)。活检细胞接种至96孔培养板,培养4小时,细胞开始贴壁,2天后细胞伸展为长梭形,3-5天可见明显增殖,7-9天可达到半融合状态。但此后细胞即出现衰退迹象,并逐渐死亡。培养细胞vWF抗体免疫荧光法鉴定呈阳性。2、冠心病患者冠状动脉病变程度及冠状动脉内皮细胞氧化损伤与血清AGEs的关系:患者Gensini总积分为56.5±41.1分,ELISA试剂盒测定血清AGEs浓度为62.3±14.9 U/mL,二者呈明显的正相关(γ=0.416,P<0.01);DCFH-DA进入活检冠状动脉内皮细胞后,被ROS氧化,生成发荧光的DCF,荧光显微镜下观察细胞内分布有弥漫的绿色荧光,分析其荧光强度为64.6±13.7,与血清AGEs呈明显的正相关(γ=0.588,P<0.01);冠状静脉窦血清NO浓度为57.0±5.6μmol/L,与血清AGEs呈明显的负相关(γ=-0.608,P<0.01)。3、AGEs对血管内皮细胞的氧化损伤效应:未加AGEs刺激时HUVECs细胞内可以检测到有一定量的ROS,表明在基础状态下,细胞内亦有ROS的生成。给予AGEs刺激后细胞内ROS明显增加,并且随AGEs浓度和孵育时间的增加而增加,以600μg/mL孵育16 h增加最明显。流式细胞仪检测结果显示,未加任何刺激剂的HUVECs经抗ICAM-1染色后其荧光检测峰较同型对照抗体染色的荧光检测峰明显右偏,其特异性抗体染色的平均荧光强度与同型对照抗体染色的平均荧光强度之比值为32.6,表明内皮细胞有ICAM-1的基础表达。经AGEs刺激后ICAM-1的表达峰明显上调,可以达到基础水平的4.5倍,表明内皮细胞受到损伤发生活化。4、各种氧化酶在AGEs诱导血管内皮细胞ROS生成中的作用大小比较:NADPH氧化酶抑制剂DPI几乎完全抑制了AGEs刺激下细胞内ROS的生成,线粒体呼吸链酶复合体I抑制剂鱼藤酮、线粒体呼吸链酶复合体II抑制剂TTFA、线粒体呼吸链酶复合体III抑制剂抗霉素A、黄嘌呤氧化酶抑制剂别嘌醇对ROS生成无明显影响,而一氧化氮合酶抑制剂L-NAME反而引起细胞内ROS的轻度增加。5、Nox4在AGEs致内皮细胞氧化损伤中的作用:RT-PCR、免疫荧光染色及Western blot检测表明,未加AGEs刺激的HUVECs有Nox4 mRNA和蛋白表达,AGEs刺激后Nox4 mRNA和蛋白表达明显增加,而Nox4 Pre-designed siRNA转染后能显著降低内皮细胞Nox4 mRNA和蛋白表达,同时细胞内ROS生成和细胞表面ICAM-1表达显著降低。结论1、通过常规的冠脉介入操作,结合免疫磁珠分选,可以从病变血管获得一定数量的有活性的人冠状动脉内皮细胞,所得细胞可用于进一步的免疫细胞化学及分子生物学检测,能够准确的反映冠状动脉内皮细胞在不同疾病状态下的功能变化,有助于深入探讨冠脉疾病的病理过程。2、血清AGEs浓度与冠状动脉病变程度及冠状动脉内皮细胞ROS水平正相关,与冠状静脉窦血清NO浓度负相关,提示AGEs可能通过内皮细胞氧化损伤途径参与动脉粥样硬化的发生和发展。3、通过培养细胞证实,AGEs与血管内皮细胞ROS生成之间存在着明确的量效关系,并导致血管内皮细胞活化。4、AGEs引起血管内皮细胞ROS水平增高主要通过NADPH氧化酶途径。5、Nox4作为内皮细胞NADPH氧化酶的主要表现形式,在AGEs诱导内皮细胞氧化损伤过程中起主要调控作用。结合其在内皮细胞分布的高度组织特异性,Nox4可能是内皮细胞ROS生成过程中发挥调控作用的关键靶点。更多还原

【Abstract】 BACKGROUNDThere is a large body of evidence linking advanced glycation end products (AGEs) to an increased risk of coronary artery disease. The main pathological mechanism by which AGEs may contribute to the development and progression of atherosclerosis is the induction of intracellular reactive oxygen species (ROS). ROS can then damage the endothelial cells (ECs). This will cause endothelial dysfunction. A more specific alteration in endothelial function that is also implicated in the pathophysiology of several conditions is endothelial activation, which refers to regulated changes in endothelial phenotype characterized by the expression of cell-surface adhesion molecules and other proteins involved in cell–cell interactions. Therefore inhibition of the generation of ROS will effectively reduce the oxidative injury to ECs and against the pathological potency of AGEs in atherosclerosis.Several potential sources of ROS are implicated in endothelial physiology and pathophysiology, including the mitochondrial electron transport chain, xanthine oxidase, cytochrome P-450 enzyme, uncoupled nitric oxide synthase (NOS), the phagocytic myeloperoxidase system and NADPH oxidase. Recent studies indicated that NADPH oxidase was the most important one for the generation of ROS in ECs by numbers of stimuli. It is unknown whether the increasing intracellular ROS in ECs by AGEs is also generated through NADPH oxidase. This is one of the objectives in our research.Once the highly specific regulating role of the NADPH oxidase could be observed, it will become an effective target point for blocking to reduce the EC injury by AGEs. But this may result in many untoward reactions, because NADPH oxidase has a wide distribution in the body, especially in the phagocytes where NADPH oxidase plays an essential role in non-specific host defence against microbial organisms. Systemic blockage of this oxidase will then lead to a disaster. How to inhibit the endothelial NADPH oxidase specifically? Broadly comparable enzymes have been reported to exist in numerous non-phagocytic cell types, such as ECs, smooth muscle cells, cardiomyocytes and fibroblasts. The molecular composition of these non-phagocytic enzymes has begun to be clarified in the last decade. They are several homologues of the gp91phox catalytic subunit. These homologues are now designated Noxs (NADPH oxidases), with gp91phox now also called Nox2. Other members of the Nox family comprise Nox1, Nox3, Nox4 and Nox5, as well as larger and more complex homologues termed Duox1 and Duox2. Their distribution appears to be highly tissue specific. Nox4 has been identified as the predominant catalytic component of endothelial NADPH oxidase. But its precise pathophysiological function remains unknown.The importance of oxidative stress in the development of endothelial activation and dysfunction is now well recognized, as is the long-term significance for future cardiovascular morbidity and mortality. However, this knowledge has so far not resulted in the introduction of new therapies. Clinical trials that have tested various antioxidants (e.g. vitamin C, vitamin E and the carotenoids) for the prevention of cardiovascular end points have generally been unsuccessful. With a better understanding of the roles that oxidative stress may play in disease pathogenesis, it seems likely that a therapeutic approach based on blanket scavenging of free radicals is probably flawed. Instead, it may be more appropriate to focus on the inhibition of specific ROS-generating enzymes. The NADPH oxidases may be especially important in this regard in view of their highly specific regulation and involvement in ROS production.OBJECTIVES1. To developed a method to isolate human coronary artery ECs in vivo from patients. These cells may be used for subsequent cellular functional analyses and help to understand mechanisms of coronary artery diseases.2. To analysis the relationships between the serum concentrations of AGEs and the severity of coronary artery lesions or oxidative dysfunction of coronary artery ECs.3. To observe the dose and time effects of AGEs on the oxidative injury to vascular ECs.4. To evaluate the contributions of several oxidases in AGEs induced oxidative injury to vascular ECs and clarify which one is the key regulator.5. To investigate the role of Nox4 in the generation of ROS and the activation of ECs by AGEs.METHODS1. Isolation and characterization of human coronary artery ECs in vivo from patients. Coronary guide wires were collected to obtain ECs samples from coronary arteries in patients undergoing percutaneous coronary interventions. Cells were eluted from wires tips and purified by immunomagnetic beads. von Willebrand factor (vWF) and CD31 were used as immunocytochemical markers to identify cells as endothelium. Cell viability was evaluated as following: (1) A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) was performed to assess the membrane integrity; (2) The uptake of fluorescent DiI-labeled acetylated low-density lipoprotein was performed to test the metabolic function; (3) Double staining with FITC-labeled annexin V and PI was used to detect apoptosis. We also attempted to culture the isolated ECs in vitro.2. Analysis the relationships between the serum concentrations of AGEs and the severity of coronary artery lesions or oxidative dysfunction of coronary artery ECs. The coronary artery lesions were quantified according to Gensini’s scoring system. The concentrations of serum AGEs were measured by an enzyme linked immunosorbent assay (ELISA) kit. Intracellular ROS of isolated coronary artery ECs were evaluated by 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). Blood samples from coronary sinus were obtained and the concentrations of serum nitric oxide (NO) were measured by NO assay kit to detect the endothelial function. Then the correlations were assessed among them.3. Dose and time effects of AGEs on the oxidative injury to vascular ECs. AGEs were prepared. Human umbilical vein endothelial cells (HUVECs) were cultured and identified by vWF. HUVECs were treated with different concentration of AGEs (100μg/mL, 200μg/mL, 300μg/mL, 400μg/mL, 500μg/mL, 600μg/mL, 700μg/mL and 800μg/mL) for different time course (1h, 2h, 4h, 8h, 16h and 24h). Intracellular ROS were evaluated by DCFH-DA and the expression of intercellular adhesion molecule-1 (ICAM-1) was determined by flow cytometric analysis.4. Evaluate the contributions of several oxidases in AGEs induced oxidative injury to vascular ECs and clarify which one is the key regulator. We used following inhibitors of oxidases: rotenone (a selective inhibitor of mitochondrial complex I), thenoyltrifluoroacetone (TTFA, a selective inhibitor of mitochondrial complex II), antimycin A (a selective inhibitor of mitochondrial complex III), allopurinol (a selective inhibitor of xanthine oxidase), Nω-Nitro-L-arginine methyl ester (L-NAME, a selective inhibitor of nitric oxide synthase), and diphenylene iodonium (DPI, a selective inhibitor of NADPH oxidase). Inhibitors were added to HUVECs 30 minutes before addition of AGEs and remained present throughout AGEs (600μg/mL) incubation. Intracellular ROS were evaluated by DCFH-DA after 16h of incubation.5. The role of Nox4 in the generation of ROS and the activation of ECs by AGEs. Nox4 mRNA expression was detected by RT-PCR, while immunofluorescence staining and western blot were performed for the assessment of Nox4 protein. Nox4 Pre-designed siRNA was transfected into vascular ECs through Lipofectamine 2000 to silence Nox4. The intracellular ROS generation and membrane ICAM-1 expression under the stimulation of AGEs with or without Nox4 Pre-designed siRNA tranfection were detected.RESULTS1. Isolation and characterization of human coronary artery ECs in vivo from patients. 37 coronary guide wires were collected to sample human coronary artery ECs in the selected 37 patients. After Wright staining, cells isolated by immunomagnetic beads displayed a round or oval morphology, more than 20μm in diameter, with a granular pink cytoplasm and an oval red-purple nucleus. The cells count per slide averaged 9.6 (range of 3–14, estimated total of 96 ECs per subject). All the cells showed expression of vWF and CD31 antigens. After FDA-PI double staining, viable cells fluoresced bright green, while nonviable cells showed bright red nuclei. Viable cells also displayed the ability to uptake DiI-labeled acetylated low-density lipoprotein. Of the total cells, about 96% showed good membrane integrity and metabolic activity. After stained by FITC-labeled annexin V and PI, a small numbers of cells exhibited an early apoptotic labeling pattern (annexin V–positive but PI–negative, about 7%) as well as a late apoptotic (or necrotic) labeling pattern (annexin V–positive and PI–positive, about 2%). Cells were plated into a single well of a 96 well plate for culture. After 4 h of incubation, the primary cells began to attach. Two days later these cells changed to a cobblestone morphology. Cells started to proliferate after 3–5 days and became a semiconfluent monolayer after 7–9 days in cultur. After this, most cells began to die gradually. The cells cultured showed positive staining for von Willebrand factor. 2. Analysis the relationships between the serum concentrations of AGEs and the severity of coronary artery lesions or oxidative dysfunction of coronary artery ECs. Gensini’s scores of Patients were 56.5±41.1. The concentrations of serum AGEs were 62.3±14.9 U/mL. The concentrations of serum AGEs significantly correlated with the severity of coronary artery lesions (γ=0.416,P<0.01). Intracellular ROS fluorescence intensities of isolated coronary artery ECs were 64.6±13.7 and significantly correlated with the concentrations of serum AGEs (γ=0.588,P<0.01). The concentrations of serum NO in coronary sinus blood samples were 57.0±5.6μmol/L and significantly negatively correlated with the concentrations of serum AGEs (γ=-0.608,P<0.01).3. Dose and time effects of AGEs on the oxidative injury to vascular ECs. Intracellular ROS had a baseline production in cultured HUVECs and were significantly increased after AGEs treatment in a concentration-dependent manner. Significant increase in ROS was observed after 2 hours of incubation and reaching a plateau at 16 hours, remaining stable thereafter. EC membrane ICAM-1 also had a baseline expression and significantly increased after AGEs stimulation. It could reach about 4.5 folds of the baseline expression.4. Evaluate the contributions of several oxidases in AGEs induced oxidative injury to vascular ECs and clarify which one is the key regulator. DPI almost completely inhibited the generation of ROS. No significant effect was observed in rotenone, TTFA, antimycin A or allopurinol. While L-NAME increased the ROS level slightly.5. The role of Nox4 in the generation of ROS and the activation of ECs by AGEs. Nox4 mRNA and protein was detected in HUVECs. AGEs stimulation caused a significantly increasing of Nox4 mRNA and protein expression. Nox4 Pre-designed siRNA transfection significantly decreased the expression of both Nox4 mRNA and Nox4 protein. At the same time, intracellular ROS generation and membrane ICAM-1 expression were both decreased.CONCLUSIONS1. Viable human coronary ECs could be obtained by guide wires combined with immunomagnetic beads during routine procedures of percutaneous coronary interventions. These cells may be used for advanced cellular functional analyses such as immunocytochemistry and molecular biology. Such information could aid in understanding mechanisms of coronary artery diseases.2. The concentrations of serum AGEs significantly correlated with the severity of coronary artery lesions and oxidative dysfunction of coronary artery ECs. This indicates that AGEs may participate in the oxidative injury to ECs and the progression of atherosclerosis.3. AGEs manifests dose and time effects on the oxidative injury to vascular ECs.4. Increasing intracellular ROS production in vascular ECs by AGEs is generated through the pathway of NADPH oxidase.5. As the predominant catalytic component of endothelial NADPH oxidase, Nox4 possesses the capability of highly specific regulation on AGEs induced ROS generation in vascular ECs. It may be a key target for blocking to reduce the EC injury.更多还原