【作者】 刘亚洋；

【导师】 吴宗贵；

【作者基本信息】 第二军医大学 ， 内科学， 2009， 博士

【摘要】 血管外膜通过直接或间接作用对血管的结构和功能调节起着重要的调控作用。血管外膜成纤维细胞(AF)是血管外膜的主要细胞成分,在动脉粥样硬化发生发展、不良血管重塑和血管再狭窄起重要作用。晚期糖基化终产物(AGEs)与AF参与糖尿病相关并发症,组织AGEs浓度与动脉粥硬化斑块的严重程度相关,AGEs的直接作用以及其与受体结合后的效应,均可引起致动脉粥样硬化相关细胞以及多种因子的激活。糖尿病患者及老年人群血管外膜中AGEs逐渐堆积,对AF等外膜细胞产生影响,但其内部机制仍不甚了解,本研究从AGEs对AF细胞迁移、氧化应激及相关炎症反应方面进行探讨。主要内容分五部分:第一部分晚期糖基化终产物对血管外膜成纤维细胞糖基化终产物受体表达的影响目的:探讨AGEs对AF晚期糖基化终产物受体(Receptor for Advanced Glycation Endproducts,RAGE)表达的影响。方法:用组织贴块法培养SD大鼠的AF。荧光免疫细胞化学鉴定细胞。细胞同步化处理,分别加入不同浓度(50、100、150、200、300μg/ml)的晚期糖基化人血清白蛋白(Advanced Glycation Endproducts-Human Serum Albumin, AGE-HSA)干预24小时,以200μg/ml人血清白蛋白(Human Serum Albumin,HSA)作为对照。用RT-PCR和Western-blot技术检测RAGE基因和蛋白的表达。结果: 50、100、150、200、300μg/ml的AGE-HSA可分别上调RAGE mRNA和蛋白的表达,且RAGE表达150μg/ml、200μg/ml、300μg/ml组与对照组差异显著(P<0.05),200μg/ml时达到峰值(P<0.05)。该效应可被p38拮抗剂、ERK1/2拮抗剂、JNK拮抗剂、坎地沙坦抑制。结论:AGEs能够上调AFs RAGE的表达,坎地沙坦能够抑制该效应。第二部分晚期糖基化终产物促进血管外膜成纤维细胞迁移机制及坎地沙坦的干预作用研究目的:观察AGEs对AF迁移的影响,探讨其机制及观察坎地沙坦干预作用。方法:用组织贴块法培养SD大鼠的AF,用transwell小室检测AGE-HSA对AF迁移的影响。用免疫印迹技术观察AGE-HSA对AF丝裂原活化蛋白激酶(MAPK)通路磷酸化影响。结果: AGE-HSA可促进p38、ERK1/2、JNK磷酸化,JNK在20min,p38、ERK1/2在30min达峰值(P<0.05)。p38拮抗剂、ERK1/2拮抗剂、JNK拮抗剂分别抑制p38、ERK1/2、JNK磷酸化,RAGE中和抗体、坎地沙坦可抑制p38、ERK1/2、JNK磷酸化。不同浓度AGE-HSA(50,100,150,200,300μg/ml)可促进血管外膜成纤维细胞的迁移,200μg/ml时达到峰值(P<0.05)。RAGE中和抗体、p38拮抗剂、ERK1/2拮抗剂、坎地沙坦均可抑制AGE所致成纤维细胞的迁移(P<0.01)。结论:AGEs经由RAGE影响MAPK通路促进血管外膜成纤维细胞迁移,坎地沙坦可通过阻断此途径抑制血管外膜成纤维细胞迁移,这可能是其血管保护作用的新机制。第三部分晚期糖基化终产物对血管外膜成纤维细胞氧化应激激活机制的研究目的:观察AGEs对AF氧化应激的影响,探讨其作用机制。方法:用组织贴块法培养SD大鼠的AF,用DCFH-DA检测AGE-HSA对AF ROS表达的影响。用RT-PCR、Western-blot检测NADPH氧化酶亚基p22phox、p47phox表达。结果: AGE-HSA可分别从基因和蛋白水平上调p22phox、p47phox的表达。氧化酶抑制剂DPI(diphenyliodonium)、APO(apocynin)、抗RAGE中和抗体、坎地沙坦可以抑制由AGE-HSA刺激引起的p22phox、p47phox表达上调(P<0.05)。AGE-HSA可促进ROS的表达上调。氧化酶抑制剂DPI、APO、抗RAGE中和抗体、坎地沙坦可以抑制由AGE-HSA刺激引起ROS的表达上调。结论:AGE-HSA经由RAGE影响ROS与p22phox、p47phox的表达上调,ROS的上调与p22phox、p47phox表达相关。NADPH氧化酶抑制剂及坎地沙坦可通过下调p22phox、p47phox的表达减少AF内ROS的产生。第四部分晚期糖基化终产物促进血管外膜成纤维细胞炎症反应机制及坎地沙坦干预研究目的:观察AGEs对AF炎症反应影响的机制及坎地沙坦干预作用。方法:用组织贴块法培养SD大鼠的AF, RT-PCR检测MCP-1、IL-6、VCAM-1 mRNA表达,ELISA检测培养液上清MCP-1、IL-6、VCAM-1。Western-blot及EMSA检测NF-κB和I-κB-α。结果:AGE-HSA 200μg/ml作用AF后0.5h、1h、2h核内NF-κB转录活性增强,作用后0.5h、1h I-κB-α磷酸化。AGE-HSA 200μg/ml处理促进NF-κB核内移,加用坎地沙坦可抑制NF-κB核内移,I-κB-α磷酸化也可被坎地沙坦抑制。不同浓度AGE-HSA(50、100、200、300μg/ml)均可使AF IL-6、VCAM-1、MCP-1 mRNA表达上调,50、100μg/ml与对照组比较mRNA表达量有显著差异(P<0.05),200、300μg/ml与对照组比较mRNA表达量有显著差异(P<0.01)。用RAGE中和抗体、NF-κB抑制剂MG-132、坎地沙坦预处理可减少由AGE-HSA所致IL-6、VCAM-1、MCP-1 mRNA表达及上清中蛋白的浓度(P<0.05)。结论:AGE-HSA通过RAGE、NF-κB途径促使炎症因子表达上调。坎地沙坦可有效抑制I-κB-α磷酸化、NF-κB核内移、炎症因子表达,提示其具有糖尿病血管并发症的治疗作用。第五部分血管外膜成纤维细胞的galectin-3表达及galectin-3基因的RNA干扰目的:观察AF半乳糖凝集素-3(galectin-3)表达,构建galectin-3 RNA干扰慢病毒载体,观察AGEs受体galectin-3对血管成纤维细胞功能影响。方法:RT-PCR检测galectin-3表达,设计RNA干扰靶点序列,合成后galectin-3短发夹RNA克隆到慢病毒载体,限制性内切酶分析和测序鉴定重组载体。工作载体和包装质粒共转染293T细胞,72小时后收集慢病毒,加入培养的血管成纤维细胞。Western blot检测半乳糖凝集素-3表达。结果:50、100、150、200、300μg/ml的AGE-HSA可分别从基因和蛋白水平上调galectin-3的表达。筛选出galectin-3的RNAi有效靶点,成功构建了galectin-3小RNA干扰的慢病毒载体,该载体能有效转染AFs,目的蛋白galectin-3达到有效敲减。结论:构建的慢病毒载体可有效抑制外膜成纤维细胞galectin-3的表达。更多还原

【Abstract】 There is a growing body of evidence that the vascular adventitia could act as a critical regulator modulating (directly or indirectly) the structure and function of the vascular wall, thus play a role in the process of atherosclerosis. As the principal cell type in the adventitia, fibroblasts were shown to correlate with vascular dysfunction, vascular restenosis and atherosclerosis. Advanced glycation end products (AGEs) and vascular adventitial fibroblasts (AFs) are involved in diabetes-related vascular complications. However, the effect of AGEs on AFs remains unclear. The aim of this study was to observe the impact of AGEs on cell migration capacity,oxidative stress and associated inflammatory responses of AFs. It is made up of four parts:Part I Effect of advanced glycation end products on the expression of receptor for advanced glycation end products in vascular adventitial fibroblastsObjective: To investigate the effect of advanced glycation end products (AGEs) on the expression of receptor for advanced glycation end products (RAGE) in vascular adventitial fibroblasts (AFs).Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. The expressions of specific antigens on cell surface were analyzed by fluorescence immunocytochemistry. After 24 hours synchronization, AFs were exposed to AGE-HSA (concentration of 0, HSA200, 50, 100, 150, 200, 300μg/ml) for 24 hours. Expressions of RAGE were measured by RT-PCR and Western-blot.Results: AGEs (concentration range from 0 to 300μg/ml) up-regulated the expression of RAGE at mRNA and protein levels, peaked at a concentration of 200μg/mL(P<0.05), this effect could be significantly inhibited by p38, ERK1/2 and JNK MAPK inhibitors as well as Candesartan.Conclusion: AGEs can up-regulate the expression of RAGE in AFs. Candesartan can inhibit this effectPart II Advanced glycation end products enhance migration of vascular adventitial fibroblasts and effect of Candesartan Objective: To investigate the effects, mechanism on the migration of rat adventitial fibroblasts induced by AGEs and to study the intervention effects of Candesartan.Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. Migratory potential was estimated by transwell chamber in vitro. Expressions of phosphorylated mitogen-activated protein kinase (MAPK) of AFs were measured by RT-PCR and Western-blot.Results: AGEs increased expression of phosphorylated forms of p38, ERK1/2 and JNK mitogen-activated protein kinases in AFs, JNK peaked at 20min and p38, ERK1/2 peaked at 30min(P<0.05). Phosphorylation of p38, ERK1/2 and JNK was suppressed by p38, ERK1/2 and JNK MAPK inhibitor,respectively.This effect also could be significantly inhibited by Candesartan and anti-RAGE neutralizing antibody. The migration of AFs enhanced by AGEs (concentration ranging from 0 to 300μg/ml) was markedly increased in a dose-dependent manner, peaked at a concentration of 200μg/mL(P<0.05). Anti-RAGE neutralizing antibody, p38, ERK1/2 MAPK inhibitors and Candesartan suppressed AGEs-induced migration of AFs(P<0.01).Conclusion: This study indicated that AGEs promote AFs migration via RAGE and MAPK pathway.Candesartan can effectively inhibit the migration of AFs. It might be a novel mechanism on vascular protection of Candesartan.Part III Mechanism on activation of oxidative stress in vascular adventitial fibroblasts induced by advanced glycation end productsObjective: To investigate the effect and mechanism of high advanced glycation end products (AGEs) on the oxidative stress in vascular adventitial fibroblasts (AFs).Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. 2,7-dichlorofluorescein-diacetate(DCFH-DA) was used as a reactive oxygen species (ROS) capture agent.The fluorescent intensity of 2,7-dichlorofluorescein (DCF), which was the product of cellular oxidation of DCFH-DA, was detected, and the level of ROS was thus measured . The expressions of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox and p47phox were measured by RT-PCR and Western-blot.Results: (1) The increase in protein and mRNA expressions of p22phox and p47phox induced by AGE-HSA was significantly inhibited by co-treatment with NADPH oxidase inhibitor APO, DPI, anti-RAGE neutralizing antibody and Candesartan (P<0.05). (2)The production of ROS in AFs induced by AGE-HSA significantly increased. The effect can be inhibited by NADPH oxidase inhibitor DPI, APO, anti-RAGE neutralizing antibody and Candesartan.Conclusion: The production of ROS in AFs is correlated with the interaction of AGEs and RAGE which can upregulate p22phox mRNA expression. NADPH oxidase inhibitors and Candesartan can reduce ROS by downregulating p22phox and p47phox expression.Part IV Advanced Glycation End Products Enhance inflammatory response of Vascular Adventitial Fibroblasts and Effect of CandesartanObjective: To investigate the effects, mechanism on the inflammation of vascular adventitial fibroblasts induced by AGEs and the intervention effects of Candesartan.Methods: Isolated vascular adventitial fibroblast of Sprague-Dawley (SD) rats were cultured. Expression of MCP-1, IL-6, VCAM-1 mRNA was analyzed by RT-PCR.MCP-1, IL-6, VCAM-1 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA). NF-κB and I-κB-αwas analyzed by electrophoretic mobility shift assay (EMSA) and Western-blot..Results: NF-κB transcriptional activity was increased after treatment with AGE-HSA (200μg/ml) for 0.5h, 1 h and 2 h. Incubating AFs with AGE-HSA (200μg/ml) for 0.5 h and for 1 h caused phosphorylation of I-κB-α. AGE-HSA (200μg/ml) treatment increased nuclear NF-κB, Candesartan co-treatment inhibited this translocation. I-κB-αphosphorylation in response to AGE-HSA (200μg/ml) was also suppressed by Candesartan. AGEs (concentration range from 0 to 300μg/ml) elevated the mRNA expression of MCP-1, IL-6, and VCAM-1. Compared with control, the expression of IL-6、VCAM-1、MCP-1 mRNA increased significantly in AGE-HSA 50,100μg/ml groups(P<0.05) and AGE-HSA 200,300μg/ml groups(P<0.01). Pretreatment of the cells with anti-RAGE neutralizing antibody , MG-132, an inhibitor of NF-κB, and Candesartan decreased the AGE-HSA induced expression of IL-6,VCAM-1, and MCP-1 and concentration of them in the supernatants(P<0.05). Conclusion: This study indicated that AGEs increase the expression of MCP-1, IL-6, and VCAM-1 in AFs via RAGE and NF-κB pathways. Candesartan can effectively inhibit phosphorylation of I-κB-α, nuclear translocation of NF-κB and expression of inflammatory factors, suggesting its treatment function of vascular complication in the diabetes.Part V Expression of Galectin-3 in Vascular Adventitial Fibroblasts and RNA Interference of Galectin-3 GeneObjective: To observe the expression of galectin-3 in AFs, to construct a lentivirus vector of RNA interference (RNAi) of galectin-3 gene and to observe effects of AGEs receptor galectin-3 on vascular fibroblasts.Methods: The expression of galectin-3 in AFs was detected by RT-PCR. DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized.Short hairpin RNA targeting to galectin-3 was cloned to lentivirus work vector.The recombined vector was identified by restriction enzyme analysis and DNA sequencing . Work vector and package plasmids were cotransfected to 293T cells.Lentivirus was collected after 72 h and was added to cultured AFs.The expression of galectin-3 was detected by Western blot.Results: AGEs (concentration range from 0 to 300μg/ml) up-regulated the expression of RAGE at mRNA and protein levels. The effective target of galectin-3 gene for RNAi was screened out. The galectin-3 siRNA lentivirus vector was established successfully. This vector can infected vascular adventitial fibroblasts and knocked down expression of galectin-3. Expression level of galectin-3 in vascular adventitial fibroblasts decreased.Conclusion: The constructed lentiviral vector can effectively inhibit the expression of galectin-3 in the adventitial fibroblasts.更多还原