【作者】 张桂林；

【导师】 张训； 侯凡凡；

【作者基本信息】 中国人民解放军第一军医大学 ， 内科学， 2003， 博士

【摘要】 背景 AGE是蛋白质、脂类的氨基与糖的醛基之间发生非酶性糖化氧化反应的终产物。随着年龄增大及血糖升高，各种组织中AGE增多。AGE形成在多种疾病如糖尿病、动脉粥样硬化、阿尔海默茨病、慢性肾衰等病理过程加速，已经证实AGE蓄积和上述疾病的发生、发展及组织病变密切相关。AGE可通过多种受体而被细胞识别，其各种生物学作用可能与其特异性受体RAGE介导有关。已有的研究表明，AGE与RAGE结合可引起细胞内氧化应激，导致核转录因子(NF)-κB的激活，诱导由NF-κB所调控的各种基因的表达。在内皮细胞，AGE可引起粘附分子(ICAM-1、VACM-1)、单核细胞趋化因子-1(MCP-1)、E-选择素等基因的表达增强。这些因子的表达可增加内皮通透性，增强炎性细胞的吸附，降低内皮细胞的正常防护功能等，故NF-κB激活引起炎症反应，导致血管内皮功能失调，可能参与AGE相关的疾病的病理进程。揭示NF-κB激活的信号通路有助于认识疾病发生发展，指导疾病的治疗。已发现，AGE引起的氧化应激，参与了NF-κB的激活。AGE引起的氧化应激与NADPH氧化酶的激活有关。NADPH氧化酶的激活可能通过两条途径，其中，佛波醇脂引起的NADPH氧化酶激活由PKC介导，而溶血卵磷脂则通过PTK介导。AGE通过何种途径激活NADPH氧化酶及激活后的NADPH氧化酶如何引起NF-κB的激活目前尚不清楚。已发现，AGE引起的ROS参与了p38 MApK的激活，对单核细胞THpl的研究发现，p38 MApK介导了NF一KB的激活。在内皮细胞，p38州叭尹K是否参与了AGE诱导的NF一KB的激活目前尚不清楚。 激活的NF一KB可诱导很多介导免疫和炎症信号的基因表达，环加氧酶一2(C0X一2)是NF一KB调节的重要蛋白之一。多种因素可诱导COX一2在内皮细胞、血管平滑肌细胞等多种细胞中的表达，引起氧化应激产生及炎症反应，参与多种疾病的病理过程。AGE可刺激内皮细胞表达多种炎症介质，作为炎症介质调节酶的COX一2是否参与AGE引起的病理生理过程目前尚不清楚。 我们以AGE修饰的人血清白蛋白(AGE一HSA)作为AGE模型，研究了AGE诱导内皮细胞NF一KB激活的信号传导机制，观察了AGE对COX一2表达的诱导作用。方法 我们用DRA病人关节组织研究NF一KB在体内的激活情况，然后选择体外培养的人脐静脉内皮细胞(HUVEC)及内皮细胞株(ECV304)为靶细胞，观察AGE修饰蛋白对内皮细胞NF.KB激活及COX一2表达的作用并对其作用机制进行探讨。 1.AGE对血管内皮细胞NF一KB的激活作用。 (1)用免疫组织化学方法检测NF一KB在DRA病人关节血管内皮细胞中的移位激活情况。 (2)用免疫组织化学法染色检测AGE处理HUVEC后，NF一KB在胞浆和胞核内的分布变化，用图象分析仪检测分析核浆灰度比值评价NF-KB的激活状况。 (3)AGE刺激ECV304引起的1кBa的变化用节触stern blot检测，NF~KB的活性用EMSA检测。 2.AGE激活血管内皮细胞NF一KB的机制。(l)用高铁细胞色素c还原法检测细胞内02一;用琉基反应试剂TbioGI。一1测定细胞内GSH。不同浓度AGE刺激ECV304，检测细胞内02一的产生量及GSH的含量变化。用抗氧化剂PDTC预处理ECV304，检测AGE诱导的02一产生量，1кBQ降解及NF~KB活性改变，观察ROS在AGE诱导的NF一KB激活中的作用。 (2)用NADPH氧化酶特异性激活抑制剂aPocynin预处理Ecv304，检测AGE诱导的02一生成量、1кBa降解及NF一KB活性改变，观察NADPH氧化酶在AGE诱导02一产生及NF一KB激活中的作用。 (3)用PKC抑制剂GF109203及PTK抑制剂Geulstein分别预处理ECV304，检测AGE诱导的02一生成量改变，观察PKC和PTK在AGE诱导NADPH氧化酶激活产生02一中的作用。 (4)用p38 MApK抑制剂SBZo358o预处理ECV304，检测A能诱导的IKB。降解及NF一KB活性改变，探讨p38MAPK在AGE诱导的NF-KB激活中的作用。 3.AGE诱导血管内皮细胞COX一2的表达及机制。 (l)用W七stem blot检测AGE诱导的内皮细胞COX一2的表达。 (2)用NF一KB抑制剂SNSO、1仗Ba抑制剂TPCK、抗氧化剂PDTC和NADPH氧化酶的激活抑制剂apoc师n预处理Ecv 304，EMsA检测NF-KB的活性，观察它们在AGE诱导的COX一2表达中的作用。结果 1.AGE可引起血管内皮细胞NF.KB的激活。(1) DRA病人关节滑膜组织中存在N下~KB的激活。免疫组化结果显示，晚期DRA病人关节滑膜中血管内皮细胞胞核NF一K BIP“染色阳性，浸润的单核细胞亦大部分胞核阳性染色，提示NF~KB被激活;早期DRA病人关节滑膜中未见明显的NF一KB激活。(2) AGE通过RAGE引起HUVECNF~KB激活。NF一KBIP65免疫组化结果显示，正常HUVEC细胞浆阳性染色，细胞核阴性;经AGE(5O林岁ml，2h)刺激后，细胞核出现阳性染色，说明AGE诱导NF一KB从细胞浆向细胞核移位，提示AGE引起NF.KB激活。用可溶性RAGE与AGE(20:1)预孵育0.5h，可阻断AGE引起的NF一KB从细胞浆向细胞核的移位。说明AGE引起的NF一KB激活是通过RAGE介导的。 (3) AGE通过诱导IKBQ降解而激活N下~KB。正常ECV304胞浆存在较高水平1кBa，AGE(50林gl]耐)刺激0.5h，1кBa降解达50%，2更多还原

【Abstract】 BackgroundAGEs are heterogeneous compounds of the end products of nonenzymatical glycation and oxidation of proteins and lipids. AGEs increase in various tissues as a function of age and hyperglycemia. The formation of AGEs is accelerated in given pathological processes and the accumulation of AGEs has been implicated in the pathogenesis of numerous disorders, including diabetes, dialysis related amyloidosis, atherosclerosis and Alzheimer’s disease. AGEs can be recognized by several cell surface receptors including scavenger receptors, p60/p90, and RAGE. RAGE is a specific receptor recognizing AGEs and has been found to be present in many cells. Interaction between RAGE and AGEs has been shown to generate an oxidative stress which trigger the activation of nuclear factor (NF)-Kfi. The activation of NF-KB may upregulate the expression of its target genes. In endothelial cells, AGEs could induce a series of gene expression such as internal cell adhesion molecular-1 (1CAM-1), vascular cell adhesion molecule-1(VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and E-selectin. Those induced factors may contribute to the increase of endothelial permeability and adhesion of inflammatory cells, which lead to dysfunction of endothelium. Therefor, activation of NF-KB and subsequent down-stream biological events might be involved in the pathogenesis ofAGEs-related diseases. Elucidating the signal transduction involved in the activation of NF-Kfi will be profitable for understanding the pathogenesis of diseases as well as for the development of novel therapeutic strategies. Oxidative stress induced by AGEs have been shown to contribute to the activation of NF-KB. Activation of NADPH oxidase has been found to be the main source of oxidative stress induced by AGEs. NADPH oxidase could be activated via two pathways. The activation of NADPH oxidase by phorbol ester (PMA) is through PKC while that by lysophosphatidylcholine (LPC) through PTk But which pathway AGEs induce the activation of NADPH oxidase via is unclear until now. In monocyte, the activation of p38 MAPK is required for the activation of NF-кB induced by AGEs. Whether p38 MAPK participates in the activation of NF-кB in endothelial cells is unknown.The activation of NF-кB could enhance expression of a series of genes relating to immunity and inflammation. Cyclooxygenase 2 is one of the important proteins regulated by NF-кB. COX-2 could be induced in endothelial cells, vascular smooth muscle cells, and some other cells. Expression of COX-2 could cause inflammation and oxidative stress which might contribute to the pathological processes. However, whether COX-2 could be induced by AGEs in endothelial cells has not been reported.The aim of the study was to investigate the intracellular mechanisms of AGEs-induced activation of NF-кB and expression of COX-2 in endothelial cells.MethodsDRA joint synovium was used as material to investigate the activationof NF-кB in vivo and human umbilical vein endothelial cells (HUVECs) and HUVEC-derived cell line (ECV304) were was used as endothelial cell models to study AGE-induced activation of NF-кB and expression of COX-2 in vitro.1. NF-кB activation induced by AGE in endothelial cells.(1) Immunohistochemical staining was used to detect the translocation of NF-кB in joint synovium of DRA patients.(2) The activation of NF-кB induced by AGEs on HUVEC was measured by immunohistochemical staining and evaluated by the ratio of optical density between nuclear and cytoplasm.(3) The degradation of IкB a and the activation of NF-кB induced by AGEs were detected by Western blot and EMSA, respectively.2. The mechanisms for AGE-induced NF-кB activation in endothelial cells.(1) Cellular O2- was measured by cytochrom C-Fe3+ reduction and GSH by ThioGlo-1 reagent. ECV304 were stimulated with different concentration of AGE, and cellular (V- and GSH were measured. To observe the role of ROS on AGE-induced NF-кB activation, ECV304 were pretreated with antioxidant PDTC, and AGE-in更多还原