【作者】 周知；

【导师】 李元建；

【作者基本信息】 中南大学 ， 药理学， 2009， 博士

【摘要】 第一章内皮祖细胞表达降钙素基因相关肽和Klotho蛋白研究背景内皮祖细胞（EPCs）是一群具有特异性归巢于血管新生组织并能分化增殖为成熟内皮细胞的干细胞,在内皮修复以及出生后血管新生中具有重要作用。大量研究表明,EPCs自分泌和旁分泌众多的细胞因子参与维持自身功能的调节。降钙素基因相关肽（CGRP）作为一种重要的内源性舒血管活性物质,参与介导多种心血管保护作用。我们和其他学者的研究发现,血管内皮也能合成和分泌CGRP,参与内皮细胞功能的调节。Klotho（K1）是另一重要内源性活性物质,具有抗衰老等生物学效应,在维持血管内皮功能方面发挥重要作用。有研究发现,重组K1蛋白可明显抑制内皮凋亡,提高内皮细胞对多种刺激因素的耐受性。鉴于EPCs是维持血管内皮完整性和促进血管新生的重要血管内皮前体细胞,且具有活跃的自分泌和旁分泌功能,本实验将分离培养人的外周和脐静脉血EPCs,以探讨其是否表达CGRP和K1蛋白。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。流式细胞仪测定早期EPCs干细胞分子标志CD133/CD34;间接免疫荧光法检测晚期EPCs内皮细胞分子标志VEGFR-2/CD31/CD144/vWF;Dil-ac-LDL和FITC-UEA-1双染鉴定正在分化的EPCs。RT-PCR检测EPCs CGRPⅠ、CGRPⅡ和K1 mRNA表达,DNA测序确证;放射免疫和ELISA分别测定EPCs CGRP和K1蛋白水平。RT-PCR测定CGRPⅠ和K1 mRNA在EPCs不同分化时间点（3 d、7d、10 d、14 d、21 d）的表达时相;激光共聚焦免疫荧光观察EPCs中CGRP和K1蛋白表达的共定位。结果（1）脐血和外周血单个核细胞用EGM-2培养可分化成EPCs,具备典型的形态学和表型特征;（2）EPCs表达CGRP（两种亚型）和K1蛋白,以CGRPⅠ和分泌型K1为主;（3）CGRPⅠ和K1 mRNA在EPCs分化中的表达时相一致,早期EPCs表达水平最高。结论EPCs（主要为早期EPCs）能合成并分泌CGRP（以CGRPⅠ为主）和Klotho（以分泌型为主）,二者之间的表达变化成正相关。第二章降钙素基因相关肽与高血压内皮祖细胞衰老研究背景动物实验与临床研究证明,原发性高血压患者和自发性高血压动物血中EPCs数量减少且功能降低,提示EPCs功能障碍可能是促进高血压发生发展的重要因素之一。进一步研究发现,高血压状态下,EPCsβ-半乳糖苷酶表达上调,端粒酶活性降低,衰老程度加剧,可能是导致EPCs功能下调的重要原因。大量研究显示,高血压时压力本身不是引起EPCs数目减少和功能障碍的直接因素,而可能与某些内源性活性物质水平异常有关,激活的肾素-血管紧张素系统（RAS）可通过氧化应激机制,加速EPCs衰老,损害其功能。近年研究证明,感觉神经的主要递质CGRP合成与释放减少与高血压的发生发展密切相关。有文献报道移植CGRP或肾上腺髓质素（CGRP超家族另一种舒血管肽）高表达的EPCs可增强其促进新生血管形成,降低肺动脉压力。基于以上报道和之前的实验发现（EPCs能合成与分泌CGRP,且CGRP的表达水平随EPCs分化成熟而下降）,我们推测高血压EPCs衰老可能与CGRP水平下降有关。EPCs衰老受多种因素影响。文献报道,K1基因缺陷小鼠除了出现与人类衰老相似表型外,其内皮依赖性的血管舒张功能降低,外周血EPCs数目明显减少,功能显著受损。由于EPCs自身表达K1基因且其表达水平随EPCs分化成熟而下降,我们推测K1基因可能参与介导高血压EPCs衰老。由于CGRP和K1在EPCs分化成熟过程中的表达变化存正相关且两者的免疫活性物质在EPCs细胞内的定位一致,本实验将探讨:（1）CGRP和Klotho与高血压EPCs衰老是否相关;（2）CGRP能否对抗高血压时由于RAS系统激活引起的EPCs衰老加速,其机制是否涉及K1。方法实验一:临床研究。分为2组:原发性高血压患者和健康对照人群各20例。取外周静脉血,测定血浆CGRP浓度（放射免疫法）和K1水平（ELISA）;分离培养EPCs,检测衰老程度（β-半乳糖苷酶染色和端粒酶定量）以及EPCs CGRP、K1 mRNA（实时定量PCR）的表达。实验二:动物实验。分为2组:自发性高血压大鼠与野生型WKY大鼠各20只。取全血,测定血浆CGRP浓度（放射免疫法）和K1水平（ELISA）;分离培养外周血来源EPCs,检测衰老程度（β-半乳糖苷酶染色和端粒酶定量）以及EPCs CGRP、K1 mRNA（实时定量PCR）的表达。实验三:体外细胞实验。采用AngⅡ诱导EPCs衰老模型,外源性给予不同剂量的CGRP或构建CGRP过表达的慢病毒感染EPCs,通过检测β-半乳糖苷酶和端粒酶活性观察CGRP抗EPCs衰老作用,并监测K1基因mRNA（实时定量PCR）和蛋白表达（Western Blot）的改变;进一步用慢病毒介导miR RNA干扰技术阻断K1表达,检测CGRP抗EPCs衰老作用是否通过调节K1蛋白生成所介导。结果（1）原发性高血压患者血浆CGRP和Klotho水平下降;外周血来源EPCsβ-半乳糖苷酶表达上调,端粒酶活性下降;EPCs CGRP和K1 mRNA表达水平下降。（2）自发性高血压大鼠血浆CGRP和Klotho水平下降;外周血来源EPCsβ-半乳糖苷酶表达上调,端粒酶活性下降;EPCs CGRP和K1 mRNA表达水平下降。（3）给予AngⅡ能使EPCsβ-半乳糖苷酶活性增加、端粒酶活性下降和Klotho分泌型蛋白表达下降;外源性给予CGRP或过表达CGRPⅠ能拈抗AngⅡ的作用,CGRP此作用可被CGRP受体阻断剂CGRP_8-37取消。（4）慢病毒介导miR干扰阻断Klotho表达能取消CGRP抗AngⅡ诱导的EPCs衰老作用。结论高血压EPCs衰老加速与体内CGRP生成减少有关,CGRP的抗EPCs的衰老作用可能通过对抗氧化应激促进抗衰老基因Klotho的表达实现。第三章吴茱萸次碱抗高血压内皮祖细胞衰老的作用和机制研究背景CGRP是辣椒素敏感感觉神经的主要递质,也是迄今发现最强的舒血管活性物质。研究报道,原发性高血压患者和自发性高血压大鼠血中CGRP浓度显著降低;α-CGRP基因敲除小鼠的基础血压增高,提示CGRP合成和释放的减少可能是高血压发生发展的重要原因之一。而且,我们前期的工作发现,高血压EPCs衰老加速与体内CGRP生成减少有关,给予外源性CGRP或CGRPⅠ过表达能有效抑制AngⅡ所致EPCs衰老,提示促进CGRP合成与分泌是抑制高血压EPCs衰老,改善EPCs功能的有效途径之一。已知辣椒素受体（VR1,又称TRPV1）是体内调节CGRP合成与释放的关键受体,在体与离体的实验均表明,激动TRPV1可促进感觉神经元合成CGRP及感觉神经末梢释放CGRP。TRPV1除广泛存在于感觉神经元胞体及其神经末梢外,也存在于内皮细胞。因此,以TRPV1为靶点寻找促进内源性CGRP的合成或释放的化合物,可能是开发抗高血压药物的新途径。吴茱萸是一种传统中药,其主要活性成分吴茱萸次碱（RUT）表现为正性肌力与正性频率,其作用可被TRPV1阻断剂Capsazepine（CAPZ）和CGRP受体阻断剂CGRP_8-37所阻断,提示其作用与激活TRPV1促进CGRP释放有关。我室新近研究证明,CGRP介导吴茱萸次碱的降压并保护血管内皮。因此,我们推测吴茱萸次碱可能通过激活TRPV1,促进CGRP合成与释放而抑制高血压动物EPCs衰老,改善EPCs功能。本实验将利用自发性高血压动物模型从整体和体外细胞水平探讨吴茱萸次碱的这一作用和机制,为吴茱萸次碱的临床应用提供新的理论和实验依据。方法实验一:动物实验。实验共分6组:野生型大鼠（WKY）组,自发性高血压大鼠（SHR）组,RUT治疗组（+RUT（10mg/kg）、+RUT（20mg/kg）和+RUT（40mg/kg））及阳性药物Losartan治疗组（+LOS（30mg/kg））。药物每天灌胃给予,连续给药2周。取全血,测定血浆CGRP浓度（放射免疫法）和K1水平（ELISA）;分离培养外周血和骨髓来源EPCs,检测衰老程度（β-半乳糖苷酶染色和TRAP-银染法）以及EPCs CGRP、K1mRNA（实时定量PCR）的表达。实验二:体外细胞实验。分离培养人脐血来源EPCs,用10^-7M AngⅡ诱导衰老,给予RUT(10^-7～10^-5M)预孵育细胞1h,48h后检测EPCs衰老程度（β-半乳糖苷酶染色和TRAP-银染法）、CGRP和K1mRNA（实时定量PCR）表达、培养液上清CGRP含量（放射免疫法）和K1分泌型蛋白表达（Western Blot）。结果（1）RUT可剂量依赖性降低自发性高血压大鼠血压,增加血浆CGRP和Klotho水平;下调大鼠外周血和骨髓来源EPCsβ-半乳糖苷酶表达,提高端粒酶活性并上调CGRP和Klotho mRNA表达。Losartan具有相似的作用。（2）EPCs表达TRPV1mRNA和蛋白。（3）给予AngⅡ能使EPCsβ-半乳糖苷酶活性增加、端粒酶活性下降和培养液上清CGRP、Klotho分泌型蛋白下降;外源性给予RUT能拮抗AngⅡ的作用,此作用可被TRPV1阻断剂CAPZ取消。结论RUT通过激活TRPV1受体,促进CGRP的合成与释放,除能有效降血压外,还能抑制EPCs衰老,其抑制EPCs衰老的机制与上调K1基因表达有关。更多还原

【Abstract】 Chapter 1Expression of calcitonin gene-related peptide and klotho in endothelial progenitor cellsBACKGROUNDEndothelial progenitor cells（EPCs）,a stem cell population,can be recruited from the bone marrow,mobilized to the circulation and differentiated into endothelial cells.There is growing evidence that many cytokines,some of which are secreted by EPCs itself,play an important role in the maintenance and regulation of EPC function.The reports from others and ours showed that endothelial cells were able to synthesize and secrete CGRP,which was supposed to participate in the protection of endothelial function.As an effective endogenous vasodilator,CGRP has been demonstrated to be involved in multiple cardiovascular protections.Klotho（K1）,a novel endogenous“anti-aging”protein,has been reported to protect against endothelial dysfunction through its effects of anti-apoptosis and anti-oxidation.Based on the evidence for an essential role of EPCs in the maintenance of endothelial function and its ability of autocrine/paracrine action,in the present study,we aimed to explore whether EPCs are able to express CGRP and K1. METHODSEPCs were isolated by density gradient centrifugation from human cord blood or peripheral 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 CD 133 and CD34 or by immunofluorescent analysis for endothelial marker proteins（vascular endothelial growth factor receptor-2[VEGFR2],CD31,CD144 and von Willebrand factor[vWF]）.The expressions of CGRPⅠ、CGRPⅡand K1 mRNA were examined by RT-PCR and verified by DNA sequence.The protein expression（both inside the cell and released into the medium） were determined by radioimmunoassay（RIA） or ELISA.We also measured the time courses of both CGRPⅠand K1 mRNA expressions at different days of EPCs cultivation（3,7,10,14 and 21）. The locations of CGRP and K1 protein inside EPCs were evaluated by laser scanning confocal microscopy.RESULTSThe present study demonstrated that:（1） Cord or peripheral blood mononuclear cells cultivated with EGM-2 generated EPCs as expected, which were characterized by typical morphology and phenotype;（2） EPCs expressed CGRP（ⅠandⅡ）,of which the predominant subtype was CGRPⅠ;EPCs also expressed klotho（including membrane and secreted forms）,and expression of the secreted form was dominated;（3） The early stage of EPCs,rather than the late ones,were the major population that produce and release CGRP and K1,and in the period of EPC differentiation,similar changes were found in the mRNA expressions of both CGRPⅠand K1.CONCLUSIONEPCs（mainly early stage of EPCs） synthesize and secrete both CGRP（mainly CGRPⅠ） and klotho（mainly the secreted form）,and the mRNA expressions of CGRPⅠand klotho are positively correlated. Chapter 2Calcitonin gene-related peptide and senescence of endothelial progenitor cells in hypertensionBACKGROUNDThere were reports that both in experimental models of hypertension and in patients with hypertension,the quantity of EPCs were reduced and their functions were impaired,suggesting that the dysfunction of EPCs are closely associated with endothelial dysfunction in hypertension. Clinical and experimental data showed that EPCs senescence was accelerated in hypertension,which resulted in the dysfunction of EPCs.It has been suggested that the alterations of internal vasoactive substances like AngⅡ,rather than elevated blood pressure itself,may account for EPCs dysfunction under hypertensive conditions.CGRP,a principal transmitter of sensory nerves,has been suggested to be strongly associated with the development of hypertension.Our previous studies showed that the synthesis and release of CGRP were significantly decreased in hypertensive patients and animals,and pharmacological induction of CGRP expression and release could lower blood pressure and reverse vascular remodeling as well as improve endothelial function.Others showed that CGRP or adrenomedullin（AM, a member of CGRP superfamily） gene transfection could enhance the therapeutic effects and animal survival in EPCs transplantation.As mentioned before,EPCs were able to express CGRP,we thus hypothesized that the secreted CGRP from EPCs,along with the circulating CGRP,might act as an autocrine/paracrine factor to regulate EPCs function,and the reduced level of CGRP might contribute to the accelerated EPCs senescence in hypertension.As an anti-aging protein with anti-oxidative property,klotho（K1） plays important role in both regulating cellular senescence and maintaining endothelial function.It has also been demonstrated that K1 knockout mice exhibit impaired ischemia-induced angiogenesis and vasculogenesis,which is associated with a decrease in EPCs number and function.We therefore speculated that the plasma level of klotho protein might be reduced in hypertension and might be associated with the accelerated senescence of EPCs.Since the alterations of CGRP and klotho in EPCs were consistent, we thus carried out in vivo and in vitro experiments to determine the relation between them and their roles in EPCs senescence under hypertensive conditions.METHODSPartⅠ:Clinical studyWe recruited 20 patients with newly diagnosed essential hypertension and 20 age-and sex-matched healthy controls.In these subjects,we took peripheral blood samples to determine plasma levels of CGRP（RIA） and klotho（ELISA）,to isolate EPCs for examining senescence-associated(β-galactosidase（SA-β-gal） and telomerase activities as well as mRNA expressions of CGRP and K1（real-time PCR）. PartⅡ:Animal experimentTwenty 16-week-old male spontaneous hypertensive rats（SHR） and 20 weight- and age-matched male Wistar-Kyoto rats（WKY/Izm） were used to collect blood for determining plasma levels of CGRP（RIA） and klotho（ELISA） and to isolate EPCs for measuring SA-β-gal and telomerase activities as well as mRNA expressions of CGRP and K1 （real-time PCR）.PartⅢ:In vitro experimentHuman cord blood samples were collected for isolation and cultivation of EPCs.The EPCs senescence model were established by incubation with AngⅡ(10_-7 M) for 48 h.The anti - senescence effects of CGRP were determined by giving exogenous CGRP(10_-8- 10^-6 M) or by over-expression of CGRP ?.EPCs senescence was evaluated by examining SA-β-gal and telomerase activities.The mRNA expressions （real-time PCR） of CGRP and K1 as well as their protein levels（RIA or western blot） were also measured.Furthermore,silence of K1 expression by miR RNA interference to determine whether the anti - senescence effect of CGRP on EPCs was through regulating K1 production.RESULTS（1） In patients with essential hypertension,the plasma levels of both CGRP and klotho were reduced;the SA-β-gal activity of peripheral blood-derived EPCs was elevated and the telomerase activity was reduced;the mRNA expressions of both CGRP and K1 in EPCs were down-regulated. （2） In spontaneous hypertensive rats（SHR）,the plasma levels of both CGRP and klotho were reduced;the SA-β-gal activity of peripheral blood-derived EPCs was elevated and the telomerase activity was reduced.The mRNA expressions of both CGRP and K1 in EPCs were down-regulated.（3） AngⅡtreatment elevated SA-β-gal activity accompanied by the reduced telomerase activity and K1 expression in EPCs,which were reversed by administration of exogenous CGRP in a concentration -dependent manner or by over-expression of CGRPⅠ.The effect of CGRP was abolished in the presence of CGRP_8-37,a selective antagonist of CGRP receptor.（4） Silence of K1 expression by miR RNAi could also abolish the anti -senescence effect of CGRP on EPCs.CONCLUSIONThe accelerated EPCs senescence in hypertension is related to the reduction of CGRP,which may work as an endogenous protective substance to counteract the oxidative stress-induced EPCs senescence in hypertension through increasing the production of anti-aging protein klotho. Chapter 3Anti-senescence effects of rutaecarpine on endothelial progenitor cells in hypertension BACKGROUNDWe have demonstrated that accelerated EPCs senescence induced by the increased oxidative stress under hypertensive conditions is related to the reduction of CGRP,while administration of exogenous CGRP or CGRPⅠover-expression could effectively inhibit EPCs senescence induced by AngⅡ.It is therefore likely that increase the production of CGRP could be a novel way to improve EPCs function through inhibiting senescence in hypertension.Though CGRP exerts extensive cardiovascular actions such as relaxing vessels,protecting endothelial function and inhibiting smooth muscle cell proliferation,its inconvenience of intravenous administration and short half-life in plasma limits its therapeutic application in the hypertensive patients.To our excitement,investigators have found that some drugs can stimulate the synthesis and release of endogenous CGRP. It has been well documented that activation of vanilloid receptor subtype 1（VR1,or transient receptor potential vanilloid 1,TRPV1）,will lead to the synthesis and release of CGRP,suggesting that activation of TRPV1 might be an effective way to prevent the development of hypertension. Rutaecarpine（RUT）,a major quinazolinocarboline alkaloid isolated from Wu-Chu-Yu,has been demonstrated to exert positive inotropic and chronotropic effects on isolated guinea pig atrium,which can be abolished by TRPV1 antagonist capsazepine（CAPZ） or CGRP_8-37, indicating that RUT can stimulate the synthesis and release of CGRP via activation of TRPV1.Our recent work has shown that the depressor and vasodilator effects of rutaecarpine are related to stimulation of endogenous CGRP synthesis and release via activation of TRPV1 on the capsaicin-sensitive sensory nerves.Besides existence in the terminals and cell bodies of sensory neurons,TRPV1 was also presented in endothelial cells.Accordingly,we presumed that RUT might have an inhibitory effect on EPCs senescence in hypertension though stimulating CGRP production via TRPV1.The aim of the present study was to test the hypothesis in both spontaneous hypertensive animals and cultured EPCs.METHODSPartⅠ:Animal experimentRats were divided into 6 groups:（1） the wild-type Wistar-Kyoto rats （WKY/Izm）;（2） spontaneous hypertensive rats（SHR）;（3） +RUT（10 mg/kg/day）;（4） +RUT（20 mg/kg/day）;（5） +RUT（40 mg/kg/day）;（6） +LOS（30 mg/kg/day）,losartan as a positive drug.All rats were treated by intragastric administration for 2 weeks. Blood was collected for determination of plasma levels of CGRP （RIA） and klotho（ELISA）;EPCs were isolated from both blood and bone marrow（femur） to detect SA-β-gal and telomerase activities as well as mRNA expressions of CGRP and K1（real-time PCR）.PartⅡ:in vitro experimentHuman cord blood samples were collected for isolation and cultivation of EPCs.The EPCs senescence model were established by incubation with AngⅡ（10-7 M） for 48 h.The anti - senescence effect of RUT were determined by giving RUT(10^-7-10^-5 M) for 1 h.EPCs senescence was evaluated by examining SA-β-gal and telomerase activities,and mRNA expressions（real-time PCR） of CGRP and K1 as well as their protein levels（RIA or western blot）.RESULTS（1） In SHR,the plasma levels of both CGRP and klotho were reduced;the SA-β-gal activity of peripheral blood-derived EPCs was elevated and the telomerase activity was reduced.The mRNA expressions of both CGRP and K1 in EPCs were down-regulated.These changes were reversed by administration of RUT.（2） Both mRNA and protein of TRPV1 were expressed in EPCs.（3） AngⅡtreatment elevated SA-β-gal activity accompanied by the reduced telomerase activity and K1 expression in EPCs,which were reversed by administration of exogenous RUT in a concentration -dependent manner.The effect of RUT was abolished in the presence of CAPZ,the antagonist of TRPV1.CONCLUSIONRUT exerts anti-senescence effect on EPCs in hypertension is through stimulating the production of CGRP via TRPV1,and the underlying mechanism is related to up-regulation of K1 expression.更多还原