【作者】 肖坚；

【导师】 李元建；

【作者基本信息】 中南大学 ， 药理学， 2009， 博士

【摘要】 第一章血浆内脏脂肪素水平与动脉粥样硬化的相关性研究目的:通过测定不同动脉粥样硬化程度受试者的血浆visfatin水平与内皮功能状态,探讨在动脉粥样硬化患者中血浆visfatin水平与血管内皮功能紊乱之间关系,评估血浆visfatin水平在预测动脉粥样硬化发生发展中的意义。方法:同时接受冠脉造影和超声检测颈总动脉、肱动脉的215例患者纳入本研究,包括55名接受PCI术的急性冠状动脉综合征（ACS）患者。采用冠脉造影及颈总动脉超声和颈总动脉IMT结果评估冠状动脉粥样硬化的严重程度,采用Gensini积分系统,将入选患者分为正常对照组、轻度动脉粥样硬化组、中度动脉粥样硬化组、重度动脉粥样硬化组。所有组患者检测血浆visfatin和血清ADMA水平并同时超声检测肱动脉FMD评估血管内皮功能。结果:（1）与正常对照组比较,动脉粥样硬化组血浆visfatin水平显著升高,且严重动脉粥样硬化组高于早期和中度动脉粥样硬化组。（2）采用线性回归统计学方法证实,血浆visfatin水平与颈总动脉IMT呈正相关（r=0.158;P<0.05）;血浆visfatin水平与冠脉Gensini积分的正相关（r=0.306;P<0.01）;血浆visfatin水平与ADMA水平呈显著正相关（r=0.323;P<0.01）,而与肱动脉FMD呈负相关（r=-0.243;P<0.01）。（3）急性冠状动脉综合征（ACS）患者接受PCI术后,血浆visfatin水平和血清AMDA水平显著降低（P<0.01）。结论:动脉粥样硬化患者血浆visfatin水平升高,并能反映血管内皮功能紊乱情况和动脉粥样硬化严重程度,提示血浆visfatin水平可能与动脉粥样硬化的发生和发展密切相关,是一个新的心血管疾病预测因子。第二章内脏脂肪素对内皮细胞血管形成能力的影响及机制研究目的:内脏脂肪素（visfatin）是一种能够促进血管新生的新型脂肪细胞因子。血管内皮生长因子（VEGF）作为血管新生中非常关键的因子,在培养的内皮细胞,二甲基精氨酸一二甲胺水解酶2（DDAH₂）能够上调VEGF的表达促进血管新生。本实验研究DDAH₂在visfatin诱导的内皮细胞血管新生中的作用。方法:在培养人脐静脉内皮细胞（HUVEC）,不同浓度的重组visfatin（0.01 nM-3 nM）处理6-24 h。Tube形成实验和迁移实验评价内皮细胞血管新生能力;Real time PCR检测DDAH₂和VEGFmRNA水平;HLPC（高效液相色普仪）测定细胞内ADMA水平;Griess法测定细胞内NO水平;ELISA试剂盒检测细胞内VEGF水平;Western blot测定Akt的磷酸化和DDAH₂蛋白水平;hDDAH₂siRNA沉默内皮细胞DDAH₂的表达。结果:重组visfatin能呈浓度和时间依赖性增强内皮细胞血管形成和迁移能力;同时,visfatin能够上调DDAH₂和VEGF的mRNA和蛋白水平。在DDAH₂沉默的内皮细胞,visfatin促内皮细胞新生的作用也显著降低。Western blot结果显示,visfatin作用内皮细胞能诱导Akt磷酸化。预先使用PI3K的特异性抑制剂能抑制visfatin诱导的DDAH₂和VEGF的表达上调。结论:我们的实验证明,visfatin能够促进内皮细胞血管新生,其机制与激活PI3K/Akt,上调DDAH-VEGF通路有关。第三章内脏脂肪素对内皮祖细胞衰老的影响目的:内脏脂肪素（Visfatin）是一种新的脂肪细胞因子。最近研究发现,visfatin通过调节SIRT1水平抗内皮细胞衰老。冠心病患者内皮祖细胞（EPCs）的衰老损伤其增殖、迁移能力和脉管的形成,同时导致循环中EPC数量的降低,提示EPCs衰老在动脉粥样硬化发生、发展中起着重要的作用。因此,本实验研究visfatin对EPCs衰老的影响。方法:密度梯度离心法分离人脐血和外周血的单个核细胞,利用EGM-2细胞培养基（含EPCs分化所需各种生长因子）进行培养,诱导单个核细胞贴壁向EPCs分化。流式细胞术测定早期EPCs干细胞分子标志CD34+、vWF;Dil-ac-LDL和FITC-UEA-1双染鉴定正在分化的EPCs。不同浓度的重组visfatin（0.01nM-10nM）处理24h。Tube形成实验和迁移实验评价EPCs血管新生能力;Real time PCR检测SIRT1mRNA水平;Western blot测定SIRT1蛋白水平;端粒酶定量法检测端粒酶活性;β-半乳糖苷酶染色试剂盒检测细胞β-半乳糖苷酶活性;使用荧光染料H2DCF检测细胞内氧自由基（ROS）的生成。结果:（1）0.1nM和1nM的visfatin能增加EPCs形成管腔样结构和促进EPCs迁移,10nM的visfatin组EPCs形成管腔样结构能力和迁移能力却显著降低（与1nM visfatin组比）。（2）0.1nM和1nMvisfatin组细胞端粒酶活性和SIRT1水平明显升高（和对照组比）,而3nM和10nM visfatin组细胞端粒酶活性和SIRT1水平明显降低（与1nM visfatin组比）。（3）0.1nM visfatin组细胞β-半乳糖苷酶活性明显降低（和对照组比）,而3nM和10nM visfatin组细胞β-半乳糖苷酶活性明显升高（与0.1nM visfatin组比）。（4）visfatin能浓度依赖地增加EPCs内ROS的水平,而预先使用DPI 300μM和Allopurinol100μM能显著抑制10nM visfatinm所致EPCs端粒酶活性、SIRT1mRNA表达的降低和β-半乳糖苷酶活性的升高（P<0.01）。结论:生理浓度visfatin能抑制EPCs的衰老,病理浓度抑制其衰老作用降低,其机制可能与激活ROS的水平,引起氧化应激降低SIRT1的表达水平有关。更多还原

【Abstract】 Objective:The aim of the present study was to investigate the pathophysiological links between plasma level of visfatin and endothelial function,and evaluate the significance of visfatin in genesis and development of atherosclerosis（AS）.Methods:In the present study,215 patients including 55 patients received the PCI operations,who had undergone coronary angiography and ultrasound detection of carotid and brachial artery,were enrolled.The severities of coronary stenosis and carotid atherosclerosis were determined by coronary angiography and carotid ultrasound and carotid intima-media thickness（IMT）,respectively.Based on the results of Gensini score,these patients were classified into four groups:control（no AS）,mild AS,middle AS and severe AS groups.The flow mediated dilation（FMD） of brachial artery was also measured by color Doppler ultrasound.The plasma levels of both visfatin and asymmetric dimethylarginine（ADMA）,a new marker of endothelial dysfunction, were determined by ELISA and HPLC,respectively.Results:Plasma level of visfatin in patients with severe AS was significantly higher than those in patients with middle AS,mild AS or in the control group.We confirmed these findings by linear regression analyses,plasma level of visfatin was positively correlated with carotid IMT,serum level of ADMA and Gensini score in all enrolled patients, and was negatively correlated with the FMD of brachial artery.Especially, the serum level of ADMA and visfatin was significantly decreased after the PCI operations.Conclusions:The present data firstly showed that plasma level of visfatin,which was markedly increased in the patient with AS,was correlated negatively with endothelial function and can reflect the severity of AS.Our present findings that visfatin is closely related to the genesis and development of AS suggest visfatin may be a new predictive factor of cardiovascular disease.Objective:Visfatin,a new adipocytokine,has been reported to promote angiogenesis.Dimethylarginine dimethylaminohydrolase （DDAH）,which can upregulate vascular endothelial growth factor （VEGF） expression in endothelial cells,is thought as a novel modulator of angiogenesis.The aim of the study was to investigate the role of DDAH₂ in visfatin-induced angiogenesis in human umbilical vein endothelial cells（HUVECs）.Methods:HUVECs were incubated with different concentrations of visfatin（0.1-3 nM） for 6-24 hours.We detected the capabilities of migration and tube formation in HUVECs to judge the angiogenic effects of visfatin.Quantitative real time PCR was used to detect the expression of DDAH₂ and VEGF mRNA.The level of ADMA was measured by high-performance liquid chromatography and the level of NO was determined indirectly as the content of nitrite and nitrate using NO assay kits.The level of VEGF was detected by the ELISA kit.DDAH₂ siRNA was used to interference the expression of mRNA and protein of DDAH₂. The phosphorylation of Akt and the protein expression of DDAH₂ were determined by western blot analysis.Results:Visfatin concentration- and time-dependently enhanced cell migration and tube formation reflecting angiogenic capability of HUVECs.Moreover,visfatin upregulated the expression of DDAH₂ and VEGF（both mRNA and protein）.Angiogenic effects of visfatin were attenuated by DDAH₂ small interfering RNA Visfatin induced protein kinase B（Akt） phosphorylation and phosphoinositide 3 kinase（PI3K） inhibitors could suppress visfatin-induced upregulation of the expression ofDDAH₂ and VEGF.Conclusions:Taken together,our results suggest that PI3K/Akt-mediated upregulation of DDAH₂ expression plays a critical role in visfatin-promoted angiogenesis via regulating VEGF-dependent pathway. Objective:Visfatin is a new adipocytokine.Recent studies have shown that visfatin enables to inhibit the endothelial cells aging via upregulating the expression of SIRT1.In coronary heart disease patients, endothelial progenitor cells（EPCs） have the impairment of proliferative, migratory capacity and network formation,and the number of circulating EPCs is reduced,suggesting that EPCs plays a critical role in genesis and development of atherosclerosisMethods:EPCs were isolated by density gradient centrifugation from human cord blood mononuclear cells,and cultured in EBM-2 supplemented with EGM-2 Single-Quots.Adherent EPCs were characterized by dual staining for acetylated low-density lipoprotein （ac-LDL） and ulex europaeus agglutinin-1（UEA-1）,and by flow cytometry detecting the stem cell marker CD34+ and vWF or by immunofluorescent analysis.After incubation of EPCs with different concentrations of visfatin（0.1-10 nM） for 24 hours,we detected the capabilities of migration and tube formation in EPCs to judge the angiogenic effects of visfatin.Quantitative real time PCR was used to detect the expression of SIRT1 mRNA and western blot analysis was used to detect the protein level of SIRT1.To quantitatively analyze telomerase activity,we used a Quantitative Telomerase Detection Kit to perform telomeric repeat amplification protocol（TRAP） assay according to the supplier’s instructio.Senescent Cells Staining Kit was used to determine the senescence-associatedβ-galactosidase activity.Intracellular oxidant productions were measured by using H2DCF,a fluoresent indicator.Results:（1） 0.1 nM and 1 nM visfatin could enhance cell migration and tube formation reflecting angiogenic capability of EPCs,the increased of migration and tube formation were decreased by 10 nM visfatin（compared with 1 nM visfatin）.（2） 0.1 nM and 1 nM visfatin could upregulate the level of SIRT1 and telomerase activity（compared with control）,the level of SIRT1 and telomerase activity were decreased with 3 and 10 nM visfatin（compared with 1 nM visfatin）.（3） Senescence-associatedβ-galactosidase activity were significantly decreased with 0.1 nM visfatin（compared with control）,β-galactosidase activity were increased with 3 and 10 nM visfatin（compared with 0.1 nM visfatin）.（4） Visfatin could concentration-dependently enhance the level of ROS,pretreatment with the inhibitor of ROS production could inhibit the decrease level of SIRT1 and telomerase activity with 10 nM visfatin and the increase inβ-galactosidase activity.Conclusions:Visfatin at the physiological concentration could inhibit the EPCs aging,and the protection of EPCs aging were attenuated by the pathological concentration of visfatin via oxidative stress-mediated downregulation of the expression of SIRT1.更多还原