血管内皮祖细胞对缺血肢体血管生成的影响

目的 从正常人经粒细胞集落刺激因子(G-CSF)动员的外周血造血干细胞中分离并扩增血管内皮祖细胞(endothelial progenitor cell,EPC),观察其促血管生成作用。方法 经G-CSF动员后富集的外周血干细胞,贴壁培养法定向扩增EPC,免疫细胞化学染色和荧光染色鉴定其内皮细胞特性。流式细胞仪检测细胞表面标记物。追踪荧光标记EPC植入裸鼠后肢缺血模型后的分布,观察肢体血流灌注并计数毛细血管。结果 贴壁细胞具有多种内皮细胞特征,培养后血液细胞标记物减少,内皮细胞标记物增加(P<0.01)。EPC特异性的分布于缺血组织,增加缺血肢体血流灌注(0.85±0.09比0.63±0.12,P<0.05)和毛细血管数(15±5比9±1,P<0.05)。结论 从G-CSF动员人外周血获得的EPC有多种内皮细胞特征,参与缺血组织血管生成,改善血流灌注。     

基质细胞衍生因子-1及其受体CXCR4轴介导内皮祖细胞参与血管损伤修复的研究进展

<正>内皮祖细胞(endothelial progenitor cells,EPCs)能够参与血管损伤修复,给再生医学治疗带来了希望。Asahara等[1]从人类外周血中分离出内皮前体细胞,发现这种细胞参与缺血组织的血管新生。多项研究证实,EPCs通过分化成新生内皮细胞替代损伤的血管内皮,参与血管损伤修复[2-3]。EPCs参与血管损伤修复过程涉及EPCs动员、迁移、分化等一系列过程,期间多种因子通过多种途径参与调节此     

粒细胞集落刺激因子动员内皮祖细胞治疗大鼠急性心肌梗死的疗效观察

目的 通过对心脏在体心功能和离体心肌储备功能测定,评价粒细胞集落刺激因子(G-CSF)动员内皮祖细胞(EPCs)对心肌梗死(MI)大鼠心功能改善的作用。方法 雄性Wistar大鼠36只,随机分为对照组、MI组和G-CSF组(每组12只),MI组和G-CSF组采用冠脉左前降支结扎法制作MI模型后,分别腹腔注射生理盐水和G-CSF连续5 d。给药后第7天,球后静脉采血,测定EPCs的数目和血浆血管内皮生长因子(VEGF)及C反应蛋白(CRP)的浓度。第4周时,进行在体心功能和离体心肌储备功能的测定,对MI大鼠的心功能进行全面评价。结果 G-CSF可增加外周血中EPCs的数目,提高血浆中VEGF的浓度。同时在体心功能和离体心肌储备功能测定的结果显示,MI大鼠的心功能得到改善。结论 G-CSF能够促进心梗大鼠EPCs的动员,增强VEGF的释放,改善MI大鼠的心功能。因此,G-CSF用于EPCs动员治疗AMI在临床上是有应用前景的。     

内皮祖细胞在心血管疾病治疗中的作用

1997年,Asahara等[1]定义了从外周血中分离出来的内皮祖细胞（endothelial progenitor cells,EPCs）.EPCs在动员、迁移后亦能形成新血管,这一过程后来被称为“血管新生”.EPCs对血管新生和内皮修复的作用展现了其潜在的治疗价值,改善EPCs功能、EPCs移植、内皮捕获支架等治疗心血管疾病提示有广泛的前景. 1 EPCs概述 自从发现外周循环中的EPCs以来,研究者已经应用多种方法来识别和分离此类细胞群.然而,EPCs仍未有共识性的定义.EPCs包含着一组细胞,这些细胞在不同的发展阶段中存在,从成血管细胞到分化成熟的内皮细胞不等.     

内皮祖细胞及造血祖细胞在心血管疾病中的意义及研究进展

1 关于内皮祖细胞在心血管疾病中的研究 1.1 内皮祖细胞定义、表型特征及来源 内皮祖细胞(endothelial progenitor cells,EPCs)是一类具有特异性归巢于损伤区域并能分化增殖为成熟内皮细胞的一群干细胞.研究表明,EPCs可能来源于骨髓,在脐带血和外周血中也有存在.EPCs的表面标志尚未明确,一般认为CD34+KDR+细胞代表EPCs,而CD34(-)CD133(+)KDR(+)细胞代表更早期循环EPCs,具有更强的参与血管新生能力.1997年,Asahara等[1]首次在中分离出CD34+及ilk-l-的单个核细胞,研究证明这些细胞在体外能够参与成年动物血管的形成,具有EPCs的功能.此后,Shi等[2]也在人的骨髓、脐带血、10 ～ 15 w胎肝及外周血中分离了较高纯度的CD34(+)细胞.随后的一些研究中研究者又从异基因骨髓移植手术后的受者外周血中分离出单个核细胞,处理后观察发现具有内皮细胞特征的梭形细胞.种种实验研究表明,EPCs来源于骨髓、脐带血及外周血液等.     

人外周血内皮祖细胞的分离培养及鉴定

目的:从人外周血中分离培养内皮祖细胞(EPCs),并探讨其体外诱导培养的条件。方法:密度梯度离心法分离正常人外周血单个核细胞,置于鼠尾胶原包被的培养瓶中进行体外培养,流式细胞术和免疫荧光法检测分化细胞表面特异性抗原标记物的表达。结果:人外周血单个核细胞在鼠尾胶原包被的培养瓶中培养后呈梭形,并表达内皮细胞的特异性抗原CD34和KDR,和干/祖细胞抗原CD133。提示这些培养细胞既具有内皮细胞的表面标志和功能,又具有祖细胞特性。结论:成功从外周血中培养出EPCs。鼠尾胶原可取代EPCs培养中常用的纤维连接蛋白,是体外分离培养外周血EPCs的更节省的一种方法。     

人外周血内皮祖细胞的分离培养及鉴定

目的从人外周血中分离培养内皮祖细胞(EPCs),并探讨其体外诱导培养的条件.方法密度梯度离心法分离正常人外周血单个核细胞,置于鼠尾胶原包被的培养瓶中进行体外培养,流式细胞术和免疫荧光法检测分化细胞表面特异性抗原标记物的表达.结果人外周血单个核细胞在鼠尾胶原包被的培养瓶中培养后呈梭形,并表达内皮细胞的特异性抗原CD34和KDR,和干/祖细胞抗原CD133.提示这些培养细胞既具有内皮细胞的表面标志和功能,又具有祖细胞特性.结论成功从外周血中培养出EPCs.鼠尾胶原可取代EPCs培养中常用的纤维连接蛋白,是体外分离培养外周血EPCs的更节省的一种方法.     

大鼠外周血晚期内皮前体细胞体外培养研究

目的体外培养大鼠外周血内皮前体细胞（EPCs）,观察细胞克隆形态并进行鉴定。方法密度梯度离心法分离SD大鼠外周血单个核细胞,EGM-2培养基离体培养。免疫荧光染色鉴定细胞CD34、CD31、CD133、FLk-1、vwF细胞表面标志。Real—TimePCR检测细胞CD34、CD133、FLk-1、eNOSmRNA表达。结果细胞培养10天后可见“铺路石样细胞”和少数“紊乱生长细胞”,传代培养后经免疫荧光及Real—TimePCR鉴定“铺路石样细胞”符合晚期EPCs特点,紊乱生长细胞不表达相应标志。结论通过体外分离长时培养可从大鼠外周血获得晚期内皮前体细胞,具有内皮细胞的特征。     

内皮祖细胞在自身免疫病中的研究进展

内皮祖细胞(endothelial progenitor cells,EPCs)是一群起源于骨髓,存在于外周血,与来源于胚胎血岛细胞具有相似特征的单核细胞群.EPCs是血管内皮细胞的前体细胞,在特定的条件下分化为成熟的内皮细胞(ECs),参与成人体内的血管发生和血管再生.1997年,Asahara等[1]在外周血中首次发现EPCs的存在,因为它们在新血管形成和血管修复方面的重要作用,使之在血管相关疾病的研究中掀起了热潮.研究发现,这类细胞具有治疗多种人类疾病的潜能,主要包括心血管疾病、缺血性周围血管病、肿瘤和自身免疫病的治疗.本文对EPCs的生物学特性及其与自身免疫病的关系综述如下.     

低氧诱导因子修饰内皮祖细胞促血管新生的研究

目的观察低氧诱导因子（hypoxia inducible factor-1α,HIF-1α）转染内皮祖细胞（endothelial progenitor cells,EPCs）后,EPCs在体外的扩增及迁移功能改变及对体内缺血下肢血管新生的影响。方法在人外周血EPCs中导入人HIF-1α基因。结果转染后的EPCs中HIF-1α、血管内皮生长因子（vascular endothelial growth factor,VEGF）mRNA及蛋白表达升高,并被特异性siRNA-HIF-1α中度抑制;功能学检测示HIF-1α过表达增加了EPC在体外的分化、扩增、迁移;内皮细胞（EC）标记物CD31、VEGFR2、eNOS及VEGF、NO分泌增加;移植HIF-1α-EPCs至缺血下肢后可见外源性EPCs存在于缺血部位;HIF-1α-EPCs较对照组进一步促进体内毛细血管数目增加。结论在体外,HIF-1α转染后的EPCs扩增及迁移功能改善;在体内,可促进缺血下肢的局部血管新生。     

Angiogenic cells can be rapidly mobilized and efficiently harvested from the blood following treatment with AMD3100

Circulating endothelial progenitor cells (EPCs) are thought to contribute to angiogenesis following vascular injury, stimulating interest in their ability to mediate therapeutic angiogenesis. However, the number of EPCs in the blood is low, limiting endogenous repair, and a method to rapidly mobilize EPCs has not been reported. In this study, healthy donors were mobilized sequentially with the CXCR4 antagonist, AMD3100, and G-CSF. The number of EPCs and circulating angiogenic cells (CACs) in the blood and pheresis product was determined and the angiogenic capacity of each cell population assessed. Compared with baseline, treatment with AMD3100 or G-CSF increased the number of blood CACs 10.0-fold +/- 4.4-fold and 8.8-fold +/- 3.7-fold, respectively. The number of EPCs in the blood increased 10.2-fold +/- 3.3-fold and 21.8-fold +/- 5.4-fold, respectively. On a percell basis, CACs harvested from G-CSF-mobilized blood displayed increased in vivo angiogenic potential compared with AMD3100-mobilized CACs. Mobilized EPCs displayed a greater proliferative capacity than EPCs isolated from baseline blood. Both CACs and EPCs were efficiently harvested by leukapheresis. Cryopreserved CACs but not EPCs retained functional activity after thawing. These data show that AMD3100 is a potent and rapid mobilizer of angiogenic cells and demonstrate the feasibility of obtaining and storing large numbers of angiogenic cells by leukapheresis.     

Diverse origin and function of cells with endothelial phenotype obtained from adult human blood

Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14+ subpopulation of PBMCs but OECs developed exclusively from the CD14- fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new cell-therapeutic approaches to ischemic cardiovascular disease.     

糖基化终末产物对人外周血内皮祖细胞数量及功能的影响

目的观察糖基化终末产物(AGEs)对体外培养的人外周血内皮祖细胞(EPCs)数量及功能的影响。方法体外培养的人外周血EPCs根据不同AGEs-人血白蛋白(HSA)浓度分为对照纽、正常组(终浓度7.5 mg/L)、干预1组(终浓度1 5 mg/L)干预2组(终浓度30 mg/L)和干预3组(终浓度60 mg/L)。采用密度梯度离心法获取人外周血单个核细胞;激光共聚焦显微镜检测其对FITC标记荆豆凝集素-1的吸附和Dil-标记乙酰化低密度脂蛋白进行细胞功能鉴定;加入AGEs后,分别采用MTT法、Boyden小室测定EPCs的增殖、迁移能力;人纤维连接蛋白检测EPCs的黏附能力;用体外血管生成分析试剂盒检测不同浓度AGEs HSA作用后的EPCs成血管能力。结果高浓度AGEs可减少EPCs贴壁细胞数量(P<0.01),减弱EPCs的黏附(P<0.01)、增殖(P<0.01)、迁移(P<0.05)和成血管能力(P<0.01)。结论 AGEs可减少人外周血EPCs的数量并使用其功能受损。     

兔外周血与骨髓源性内皮祖细胞培养鉴定及生物学特性的研究

目的:探索并建立兔内皮祖细胞(EPCs)的体外培养、鉴定方法。方法:采用密度梯度分离法分别从兔外周血和骨髓中分离出单个核细胞,接种于预包被明胶的培养瓶,并应用培养基(EBM-2)诱导培养,诱导分化为EPCs;在倒置显微镜下观察两种源性细胞生长过程;台盼蓝法测定细胞传代成活率;通过绘制生长曲线法、MTT法、DNA周期检测比较两种源性获得的EPCs增殖情况;采用免疫荧光染色观察EPCs相关抗原CD34、CD31、CD133及vWF因子的表达。结果:两种源性EPCs均于培养3～4d完全贴壁,呈克隆样生长,7～9d形成类似成熟血管内皮细胞形态,细胞数量逐渐增多,细胞成活率均为95%。两种源性获得的第2代细胞生长曲线近似"s"形、MTT法显示3～5d细胞增殖较明显,外周血源性EPCs G0-G1期和S+G2+M期所占比例分别为96.48%和3.52%,骨髓源性EPCs G0-G1期和S+G2+M期所占比例分别为97.11%和1.84%。第2代EPCs进行免疫荧光鉴定结果显示:两种源性内皮祖细胞均阳性表达CD34、CD133、vWF因子,CD31弱阳性表达。结论:两种源性的EPCs均可高效获得且在体外稳定培养。与传统的骨髓源性EPCs相比较,从外周血源获得的EPCs是一种可靠、简便的培养方法,为组织工程学提供理想的细胞来源。     

流体切应力对人内皮祖细胞组织纤溶酶原激活物基因表达的影响

目的研究不同流体切应力务件下,内皮祖细胞组织纤溶酶原激活物(tissue-type plasminogen activator,t-PA)基因表达的变化。方法分离健康志愿者外周血的单个核细胞并诱导分化成内皮祖细胞,并通过相差显微镜和免疫荧光标记等方法进行形态学观察和鉴定。种植内皮祖细胞到小径人工血管表面后,分为静态组、低切应力组(0．5 Pa)、中切应力组(1．5 Pa)和高切应力组(2．5 Pa)4个不同处理组,之后用ELISA各组不同时间点培养液中t-PA的水平,并用半定量RT-PCR测定切应力处理25 h后,不同切应力组内皮祖细胞t-PA的基因表达。结果外周血单个核细胞在体外诱导分化成为内皮祖细胞,ac-LDL吞噬及lectin抗体荧光标记双阳性,FLK-1和vWF免疫荧光抗体染色均为阳性。提高流体切应力可以增加t-PA的分泌,上调内皮祖细胞t-PA的基因表达。结论提高流体切应力能明显促进内皮祖细胞t-PA的分泌和基因表达,有助于改善内皮祖细胞的生理功能。     

人体外周血来源成体内皮前体细胞在体外培养过程中的单核细胞和巨噬细胞功能表现

目的：研究成体内皮前体细胞生物学特性,尤其对该种细胞所表现的单核细胞和巨噬细胞特点进行探讨。方法：人体外周血来源的单个核细胞被接种于纤维粘连蛋白(Fibronectin)包被的培养皿中,在 EGM-2培养液中培养。描述贴壁细胞生长曲线,对细胞表型及功能特点进行检测。结果：部分单个核细胞变形成为长梭形细胞,但未表现出明显的增殖能力。这些细胞可以表达部分内皮系表面标志物及单核细胞标志物 CD14。在第4,14,28天进行的 Dil 标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)和印度墨汁双摄取实验显示这种细胞能够同时吞噬这两种物质,却没有类似于内皮细胞的体外成血管现象,从而说明这种细胞有类似于单核细胞和巨噬细胞的吞噬功能。结论：人体外周血单个核细胞来源成年内皮前体细胞在体外培养过程中表现典型的单核巨噬细胞功能,且无明显增殖能力。     

Erythropoietin regulates endothelial progenitor cells

Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neoangiogenesis, and their number in peripheral blood correlates with endothelial function and cardiovascular risk. We tested the hypothesis that the cytokine erythropoietin (EPO) stimulates EPCs in humans. We studied 11 patients with renal anemia and 4 healthy subjects who received standard doses of recombinant human EPO (rhEPO). Treatment with rhEPO caused a significant mobilization of CD34(+)/CD45(+) circulating progenitor cells in peripheral blood (measured by flow cytometry), and increased the number of functionally active EPCs (measured by in vitro assay) in patients (week 2, 312% +/- 31%; week 8, 308% +/- 40%; both P <.01 versus baseline) as well as in healthy subjects (week 8, 194% +/- 15%; P <.05 versus baseline). The effect on EPCs was already observed with an rhEPO dose of about 30 IU/kg per week. Administration of rhEPO increased the number of functionally active EPCs by differentiation in vitro in a dose-dependent manner, assessed in cell culture and by tube formation assay. Furthermore, rhEPO activates the Akt protein kinase pathway in EPCs. Erythropoietin increases the number of functionally active EPCs in humans. Administration of rhEPO or EPO analogs may open new therapeutic strategies in regenerative cardiovascular medicine.     

黄芪多糖对2型糖尿病患者外周血内皮祖细胞增殖及NO生成的影响

目的观察黄芪多糖（APS）对2型糖尿病（T2DM）患者外周血内皮祖细胞（EPCs）增殖和NO生成的影响,并探讨其机制。方法密度梯度离心法获取外周血单个核细胞,培养7 d后鉴定EPCs,检测APS对T2DM组EPCs增殖的影响。观察APS对T2DM组EPCs NO浓度影响的时效和量效关系,然后予磷脂酰肌醇三磷酸激酶（PI3K）抑制剂LY294002和APS共培养,检测NO浓度。结果 APS在一定剂量和时间范围内能促进T2DM组EPCs增殖（P〈0.05）,并呈剂量和时间依赖性地升高EPCs NO浓度;PI3K抑制剂LY294002能阻断APS升高的NO水平（P〈0.05）。结论鼠尾胶原可代替EPCs培养中常用的纤维连接蛋白,是体外分离培养外周血EPCs更节省的一种方法。APS能促进EPCs增殖,并促进其NO的生成,PI3K抑制剂LY294002能阻断APS引起的NO的生成。     

Nifedipine improves the migratory ability of circulating endothelial progenitor cells depending on manganese superoxide dismutase upregulation

BACKGROUND: Migratory ability of resident endothelial cells and their circulating progenitors, that is endothelial progenitor cells (EPCs), represent a crucial event in vascular repairing processes. Although oxidants are known to counteract the migratory ability of resident endothelial cells, their possible role in modulating EPC motility is unknown. EPCs exhibit stronger resistance to oxidants than mature endothelial cells mainly because of higher expression of manganese (Mn) superoxide dismutase (SOD). As nifedipine is a dihydropyridine calcium antagonist known to enhance MnSOD expression in mature endothelial cells, we investigated the effects of nifedipine on MnSOD expression and motility in EPCs. METHODS AND RESULTS: EPCs were isolated from peripheral blood of healthy donors and cultured in fibronectin-coated flasks. Nifedipine improved both motility of cultured EPCs (+55% vs. control, P = 0.007) and their adhesion to tumor necrosis factor-alpha-activated mature endothelial cells (+33% vs. control, P = 0.03). Reduction of EPC dichlorofluorescein content (-32% vs. control, P = 0.009) and a parallel increase in nitrite plus nitrate concentration in EPC supernatants (+25% vs. control, P = 0.009) were also observed. The study of SOD showed a nifedipine-dependent upregulation of MnSOD in a time-dependent and dose-dependent manner. MnSOD expression blockade by RNA interference abolished nifedipine effect on EPC motility. Although nifedipine also increased vascular endothelial growth factor (VEGF) release from EPCs, its effect on EPC motility was unaffected by an anti-VEGF antibody. CONCLUSION: Nifedipine improves EPC motility due to MnSOD upregulation. The capability of this dihydropyridine calcium antagonist to reduce cardiovascular events might also be related to improved EPC function.