【作者】 袁琼；

【导师】 李元建；

【作者基本信息】 中南大学 ， 药理学， 2010， 博士

【摘要】 研究背景内皮祖细胞(endothelial progenitor cells, EPCs)是一类能分化为成熟的内皮细胞及心肌细胞,促进血管新生和改善心功能的干细胞。EPCs参与血管修复和血管新生,为治疗缺血性疾病提供了新的策略。血管内皮生长因子(vascular endothelial growth factor, VEGF)是调节EPCs功能最强的因子。VEGF与2型VEGF受体(KDR)相互作用而调节EPCs功能,包括内皮修复与血管新生。二甲基精氨酸-二甲胺水解酶(dimethylarginine dimethylaminohydrolase, DDAH)能特异性的水解L-精氨酸的同类物非对称二甲基精氨酸(asymmetric dimethylarginine, ADMA)。ADMA是一种主要的内源性一氧化氮合酶(nitric oxide synthase, NOS)抑制物,能竞争性抑制NOS,减少一氧化氮(nitric oxide, NO)合成。研究发现,内皮细胞衰老和血管新生涉及DDAH/ADMA途径。人类沉默信息调节因子2(Silent information regulator 2, Sir2) SIRT1是Ⅲ型组蛋白去乙酰化蛋白,能引起基因表达沉默。研究发现,SIRT1通过DDAH/ADMA途径抑制内皮细胞衰老。文献报道,高糖诱导EPCs衰老涉及SIT1途径。基于DDAH/ADMA与SIRT1参与血管新生及细胞衰老的调节以及SIRT1介导DDAH/ADMA途径的效应,我们推测DDAH/ADMA可能通过VEGF/KDR途径参与EPCs分化与功能的调控,该作用可能涉及SIRT1途径。因此,本实验在体外培养人外周血来源EPCs研究了DDAH/ADMA对EPCs功能与衰老的影响。方法EPCs培养与鉴定：梯度离心加选择性粘附分离健康人外周血EPCs,用内皮基础培养基EBM-2培养EPCs。培养7天后鉴定,包括乙酰化低密度脂蛋白(Dil-acLDL)与荆豆凝集素1(FITC-UEA-1)双染鉴定分化EPCS;流式细胞仪检测细胞CD133、CD34、CD45、KDR、vWF等标记物表达。DDAH对EPCs分化及功能调控及机制：提取分化第7天的EPCs,检测DDAH1和DDAH2表达；提取第7和17天EPCs,检测DDAH2、SIRT1表达。为了探讨DDAH2对EPCs功能的调控,运用pGCSIL-GFP-hDDAH2慢病毒转染EPCs沉默DDAH2表达；为了探讨SIRT1对DDAH2表达的调控,运用pGCSIL-GFP-hSIRT1慢病毒转染EPCs沉默SIRT1表达。提取细胞mRNA, Real-time PCR检测DDAH1、DDAP2、SIRT1、VEGF和KDR mRNA表达；流式细胞仪和Western Blot检测DDAH2和SIRT1蛋白表达；高效液相色谱法(high performance liquid chromatography, HPLC)检测细胞上清ADMA;β-半乳糖苷酶活性评价EPCs衰老程度；tube小管形成实验评价EPCs血管形成能力；纤维连接蛋白检测EPCs粘附能力。结果人外周血EPCs主要以DDAH2亚型表达为主,且随着EPCs分化时间延长而增加。干扰DDAH2表达能诱导EPCs衰老并抑制血管形成和粘附能力,抑制VEGF/KDR的mRNA表达。SIRT1能随着EPCs分化而表达增加,干扰SIRT1表达能下调DDAH2、VEGF/KDR表达,但对细胞上清ADMA浓度没有影响。结论DDAH2参与EPCs分化过程,并通过调节VEGF/KDR途径抑制EPCs的衰老,其作用机制涉及SIRT1途径。研究背景血管病变是糖尿病主要并发症之一。临床与动物实验发现糖尿病时,EPCs衰老率显著增加、功能受损。高糖能诱导EPCs衰老、血管形成能力减弱,导致糖尿病血管功能减弱、侧枝循环建立减少,引发心血管疾病。ADMA是一种新的心血管疾病预测因子,是内源性NOS抑制物,能竞争性抑制NOS,减少NO的合成。糖尿病时,细胞DDAH2表达降低、活性减弱,引起ADMA浓度增加并抑制NO生成。临床与动物实验证明,糖尿病时EPCs功能受损与NO浓度降低有关。细胞实验证明,ADMA能直接损伤EPCs功能。糖尿病或高糖孵育EPCs时,EPCs SIRT1表达下调,同时伴随细胞衰老和血管形成功能减退。内皮细胞实验发现,SIRT1可上调DDAH2表达,增加活性,促进ADMA水解,抑制内皮细胞自然衰老过程。本实验在2型糖尿病患者与培养EPCs细胞探讨了EPCs衰老与DDAH/ADMA之间的关系,以及该过程是否涉及SIRT1途径。方法临床研究：对照组与2型糖尿病患者组各40例。取外周静脉血,分离血浆与细胞。HPLC检测血浆ADMA浓度；梯度离心加粘附分离细胞EPCs,β-半乳糖苷酶活性评价细胞衰老,Real-time PCR法检测细胞DDAH2、SIRT1 mRNA表达。细胞实验：外源性给予葡萄糖(10,20,30 mmol/L)孵育EPCs建立高糖损伤EPCs细胞模型,以甘露醇(10,20,30 mmol/L)作为渗透压对照组,探讨高糖诱导EPCs衰老；构建pGC-FU-hDDAH2慢病毒探讨DDAH2抑制高糖诱导EPCs衰老；外源性给予ADMA(0, 0.3,0.6, 1pmol/L)探讨ADMA诱导EPCs衰老作用；运用SIRT1激动剂白藜芦醇(resveratrol, RSV)探讨SIRT1调节高糖诱导细胞衰老作用机制。梯度离心加粘附分离正常人外周血EPCs。细胞处理48h后,β-半乳糖苷酶活性评价细胞衰老。其他指标的检测为细胞处理24h时,Real-time PCR与Western Blot检测DDAH2、SIRT1 mRNA与蛋白表达,HPLC检测细胞上清ADMA水平,Griess法检测上清NO水平。结果2型糖尿病患者外周血EPCs衰老率显著升高,DDAH2与SIRT1 mRNA表达下调,同时伴随ADMA水平升高。葡萄糖能浓度依赖性地诱导健康人外周血EPCs衰老,下调EPCs DDAH2 mRNA和蛋白表达,增加培养上清ADMA水平并减少NO生成。渗透压对照组对细胞没有明显影响。过表达hDDAH2能抑制高糖诱导的EPCs衰老,并降低培养液中ADMA水平。外源性ADMA能浓度依赖性的诱导EPCs衰老。高糖能下调SIRT1 mRNA和蛋白表达。SIRT1激动剂RSV能显著抑制高糖诱导EPCs衰老,并且逆转高糖所致的DDAH2 mRNA表达下调及ADMA浓度升高。结论DDAH2/ADMA参与了高糖诱导的EPCs衰老,其作用涉及SIRT1途径。研究背景白藜芦醇(resveratrol, RSV)是一种多酚类的SIRT1天然激动剂。SIRT1参与糖代谢和胰岛素分泌的调节。糖尿病大鼠实验证明,RSV能上调VEGF与eNOS表达,抑制氧化应激反应,提高糖尿病大鼠骨髓细胞血管形成能力。细胞实验证明,RSV促进人外周血EPCs增殖、迁移以及血管新生。此外,RSV也能抑制TNF-α所致EPCs损伤。(E)-3,5,4’-三甲氧基-1,2-二苯乙烯((E)-3,5,4’-trimethoxystilbene, BTM-0512)是RSV的甲基化衍生物,其口服吸收率与半衰期均优于RSV。依据RSV对血管内皮和EPC保护作用以及SIRT1-DDAH2/ADMA对EPCs功能及衰老的调节作用,本实验在高脂饮食加单次腹腔注射链脲佐菌素(Streptozotocin, STZ)诱发的2型糖尿病大鼠模型与培养大鼠骨髓EPCs细胞,探讨RSV衍生物BTM-0512能否改善胰岛素抵抗,以及对EPCs衰老的影响及机制。方法动物实验：高脂饮食加单次腹腔注射STZ (35mg/kg)建立2型糖尿病大鼠模型。实验分为正常对照组、模型组、BTM-0512低剂量组(10mg/kg)和BTM-0512高剂量组(40mg/kg)。大鼠连续灌胃3周。取大鼠全血,分离血浆检测大鼠空腹血糖(FBG)、糖化血红蛋白(Hb1AC)、葡萄糖耐量(OGTT)、HOME.IR、胰岛素敏感指数(IAI)、胰岛素分泌指数(IS)以及ADMA浓度。切取胸主动脉观测内皮依赖性舒张反应,免疫组化检测血管内皮DDAH2、SIRT1蛋白表达。采用梯度离心加选择性粘附分离大鼠骨髓EPCs细胞,β-半乳糖苷酶活性评价细胞衰老程度,Real-time PCR检测EPCs DDAH2、SIRT1 mRNA表达,tube小管形成实验检测EPCs血管形成能力,transwell小室评价EPCs迁移能力,纤维连接蛋白粘附法评价EPCs粘附能力。细胞实验：高糖处理EPCs,探讨BTM-0512 (0.3,1,3μM)抑制高糖诱导EPCs衰老及可能机制,给予SIRT1特异性阻断剂splitomicin (50μM)探讨SIRT1在BTM-0512对EPCs保护中作用。β-半乳糖苷酶活性评价细胞衰老,Real-time PCR检测DDAH2、SIRT1 mRNA表达,Western Blot检测DDAH2蛋白表达;HPLC检测细胞上清ADMA水平。结果BTM-0512能剂量依赖性降低2型糖尿病大鼠FBG和HblAC,改善HOME.IR和IAI,但对OGTT与IS没有影响。BTM-0512能剂量依赖性改善2型糖尿病大鼠血管舒张能力,抑制骨髓EPCs衰老,保护EPCs迁移、粘附及血管形成功能；BTM-0512能上调血管内皮及EPCs DDAH2、SIRT1表达。在培养EPCs, BTM-0512能剂量依赖性抑制高糖诱导细胞衰老,上调细胞DDAH2、SIRT1 mRNA表达并降低培养上清中ADMA浓度。SIRT1抑制剂splitomicin (50μmol/L)能取消BTM-0512对高糖诱导的EPCs衰老与EPCs DDAH2 mRNA表达下调,以及上清ADMA浓度升高的逆转作用。结论1.BTM-0512可降低2型糖尿病大鼠血糖,改善IR,并能改善血管舒张功能。2.BTM-0512能减轻高糖所致EPCs衰老,其机制涉及SIRT1-DDAH2/ADMA途径。更多还原

【Abstract】 Endothelial progenitor cells (EPCs), which can differentiate into mature endothelial cells and myocytes, promote neovascularization and improve cardiac function. EPCs participate in the reparation of vessles and angiogenesis, and are used for the treatment of ischemic diseases.Vascular endothelial growth factor (VEGF) is a potent factor regulating EPCs functions, including reendothelization and angiogenesis. The effects of VEGF have been shown to mediated by type 2 VEGF receptor (KDR).It is know that dimethylarginine dimethylaminohydrolase (DDAH) metabolizes asymmetric dimethylarginine (ADMA), which is endogenous inhibitor of nitric oxide synthase (NOS) activity and inhibits nitric oxide (NO) production. It has been demonstrated that the DDAH/ADMA pathway is involed in senescence of endothelial cell and angiogesis.SIRT1, human silent information regulator 2 (Sir2), is a potent NAD+-dependent protein deacetylase and plays an important role in the maintenance of gene silencing. SIRT1 inhibites senescence of endothelial cells through the DDAH2/ADMA pathway. It has been shown that SIRT1 is involved in the senescence of EPCs induced by high glucose.Based on the regulatory effect of DDAH/ADMA and SIRT1 on angiogenesis and cell senescence, and SIRT1-mediated the effect of DDAH/ADMA, therefore, in the present study we tested the effect of DDAH/ADMA and SIRT1 on senescenc and fucntion of EPCs.METHODSEPCs were isolated by density gradient centrifugation from human peripheral blood mononuclear cells, and cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 Single-Quots. On the 7th day, EPCs were characterized by dual staining for Dil-acetylated low-density lipoprotein (Dil-acLDL) and FITC-ulex europaeus agglutinin-1 (FITC-UEA-1), and by flow cell markers including CD 133, CD34, KDR, CD45 and von willebrand factor (vWF).To test the effect of DDAH on the differentiation and function of EPCs, mRNA expressions of DDAH1 and 2 were detected at 7th day, and the expressions of DDAH2 and SIRT1 mRNA and protein were detected at 7th and 17th days. To study the regulatory effect of DDAH2 on senescence and fucntion of EPCs, cells were transfected with pGCSIL-GFP-hDDAH2 shRNA to interrupt expression of DDAH2 protein. To study the regulatory effect of SIRT1 on DDAH2, cells were transfected with pGCSIL-GFP-hSIRT1 shRNA to interrupt expression of SIRT1 protein. The levels of DDAH1, DDAH2, VEGF, KDR and SIRT1 mRNA were analyzed by Real-time PCR. Protein expressions of DDAH2 and SIRT1 were detected by flow cytometry or Western Blotting. The level of ADMA was detected by high performance liquid chromatography (HPLC). Cell senescence, angiogenesis and adhesion were determined by senescence-associatedβ-galactosidase (SA-β-gal) activity assay, tube formation and fibronectin.RESULTSPeripheral blood EPCs predominantly expressed DDAH2 which was increased with EPCs differentiation. Interrupting DDAH2 expression induced EPCs senecence and dysfunction, including angiogenesis and adhesion. The expressions of VEGF and KDR mRNA were down-regulated. The expression of SIRT1 was increased with EPCs differentiation. Interrupting SIRT1 inhibited the expressions of DDAH2, VEGF and KDR, but had no effect on the level of ADMA.CONCLUSIONDDAH2 participated in the differentiation of EPCs and regulated the senescence and fucntion of EPCs through the VEGF/KDR pathway by the activation of SIRT1. Vascular disease is a main complication of diabetes mellitus. Clinical and animal researches have showed that diabetes accelareted EPCs senescence and attenuated EPCs function. And high glucose induced EPCs senescence and impaired angiogenesis, resulting in dysfunction of vessels and reduction of collateral circulation. These results suggest that EPCs senescence may be related with the development of cardiovascular diseases.ADMA, a major endogenous inhibitor of NOS, has been considered as a novel risk factor for cardiovascular diseases. It has been demonstrated that the plasma level of ADMA is elevated and NO concentration is decreased concomitantly with a reduction of expression and acitvity of DDAH in patients with diabetes. Clinical and animal researches have showed that dysfunction of EPCs is related with reduction of NO level in diabetes. Exogenous ADMA directly induced the damage of EPCs. Studies in vivo or in vitro have documented that high glucose treatment induced senescence and angiogenesis impairment of EPCs and down-regulation of SIRT1 expression. It has been reported that the regulatory effect of SIRT1 on endothelial cells senescnece is mediated by the DDAH2/ADMA pathway.In the present experiment, we explored the regulatory effect of DDAH2/ADMA on sensecence of EPCs in diabetic rats or EPCs treated with high glucose. Since SIRT1 plays an important role in the development of senescence of EPCs, we also tested the effect of SIRT1 on the DDAH2/ADMA pathway.METHODSForty control people and 40 T2DM patients were recruied, and control people were age and sex matched with T2DM patients. The level of ADMA in plasma was detected by HPLC. EPCs were isolated by density gradient centrifugation from human peripheral blood mononuclear cells. EPCs senescence was evaluated by SA-β-gal activity and the expression of DDAH2, SIRT1 mRNA was measured by Real-time PCR.EPCs were treated with glucose (10,20 or 30 mmol/L) for inducing senescence, and mannitol(10,20,30 mM) was used as osmotic pressure control. To study the effect of DDAH2 on senescence of EPCs, EPCs were transfected with pGC-FU-hDDAH2. ADMA(0,0.3,0.6,1μmol/L) was used to directly induce senscence of EPCs. The effect of resveratrol (RSV), a agonist of SIRT1, on senscence of EPCs was observed.Peripheral blood mononuclear cells were collected for isolating EPCs. EPCs senescence was evaluated by SA-β-gal activity. The expressions of DDAH2 and SIRT1 mRNA or protein were measured by Real-time PCR and by Wetern Blot, respectively. The level of ADMA was detected by HPLC, and the level of NO was detected by Griess.RESULTSIn T2DM patients, the percentage of EPCs senescence was increased, while the expressions of both DDAH2 and SIRT1 mRNA were down-regulated concomitantly with an increase of concentrations of ADMA in plasma.High glucose induced senescenc of EPCs in a dose-dependent manner. The mRNA and protein expression of DDAH2 were down-regulated, and level of NO was decreased while level of ADMA was increased in the presence of high glucose. Overexpression of DDAH2 reversed the effect of glucose, but did not affect NO concentration in cultured EPCs. ADMA induced the senescence of EPCs in a dose-dependent manner. High glucose treatment down-regulated the mRNA and protein of SIRT1, which an effect was attenuated by RSV. CONCLUSIONDDAH2/ADMA pathway participats in senescence of EPCs induced by high glucose, which is related with activation of SIRT1. Resveratrol (trans-3,5,4’-trihydroxystilbene) is a natural activator of SIRT1. Growing evidence suggests that SIRT1 regulates glucose and insulin secretion. It has been showed that in diabetic rats, RSV exerts multiple benificial effects including upregulating expression of VEGF and eNOS, inhibiting oxidative stress and mobilizing angiogenesis. In cultured EPCs, RSV can induce cell proliferation, migration and angiogenesis. Others have found that RSV reduced the injury of EPCs by TNF-a.(E)-3,5,4’-trimethoxystilbene (BTM-0512) is the methylated derivative of RSV. The absorbance rate of BTM-0512 is higher than RSV, and its half-life is extended.Based on the protective effect of RSV on senescence of endothelial cells and EPCs and the regulatory effect of SIRT1-DDAH2/ADMA pathway on EPCs function, therefore, we tested the influence of BTM-0512 on insulin resistance and senescence of EPCs in type 2 diabetic rats and cultured EPCs. METHODSType 2 diabetic rat were established by bonus injection of streptozotocin (STZ,35 mg/kg, intraperitonealy) and feeding high-fat/high-sucrose diet. Rats were divided into 4 groups:control, T2DM, T2DM plus BTM-0512(10 mg/kg) and T2DM plus BTM-0512(40 mg/kg). The animals were treated with drugs for 3 weeks.The concentrations of fasting blood glucose (FBG) and glycosylated hemoglobin (Hb1AC), the level of fast insulin (FIns) and oral glucose tolerance (OGTT) were detected. The values of HOME.IR, insulin activity index (IAI) and insulin secretion index (IS) were calculated to evaluate insulin resistance (IR). The level of ADMA was measured. Vasodilator responses to acetylcholine in aortic rings and the protein expression of expression of DDAH2 and SIRT1 in the isolated aortia were measured. EPCs were isolated by density gradient centrifugation from rat bone marrow. Cell senescence, angiogenesis, adhesion and migration were determined by SA-β-gal activity assay, tube formation, fibronectin and transwell assay respectively. The mRNA expressions of DDAH2 and SIRT1 were measured by Real-time PCR.EPCs from rat bone marrow were characterized by dual staining for Dil-acLDL and UEA-1 on the 7th day. EPCs were treated with glucose (30 mmol/L) for inducing senescence. To study the effect of BTM-0512 on senescence of EPCs, EPCs were pretreated with BTM-0512 (0-3 μmol/L). To study the role of SIRT1 in BTM-0512 effect, EPCs were pretreated with splitomicin (50μmol/L), the special inhibitor of SIRT1.EPCs senescence was evaluated by examining SA-β-gal activity. The mRNA expressions of DDAH2 and SIRT1 were measured by Real-time PCR. Protein expression was detected by Wetern Blot. The level of ADMA was detected by HPLC. The level of NO was detected by Griess.RESULTSBTM-0512 significantly decreased the level of FBG and Hb1 AC in a dose-dependent manner. BTM-0512 improved the HOME.IR and IAI, but had no effect on OGTT or IS. BTM-0512 improved vasodilator responses to acetylcholine in diabetic rats. BTM-0512 inhibited senescence of EPCs and up-regulated the expression of DDAH2 and SIRT1 protein of endothelial cells as well as the expression of DDAH2 and SIRT1 mRNA of EPCs.BTM-0512 attenuated senescence of EPCs induced by high-glucose in a dose-dependent manner. BTM-0512 also reversed high glucose induced the downregulation of SIRT1 and DDAH2 mRNA expression with increased the level of ADMA, which were reversed by splimtomicin (50μM), the sepecial inhibiotr of SIRT1. CONCLUSION1. BTM-0512 reduced fasting blood glucose and improved insulin resistance in type 2 diabetic rats. BTM-0512 also improved endothelial function in diabetic rats.2. BTM-0512 exerted the beneficial effects on high glucose induced EPCs senescence, and the effects of BTM-0512 was related to activation of SIRT1-DDAH2/ADMA pathway.更多还原