银杏叶提取物对外周血内皮祖细胞数量和功能的影响

目的观察银杏叶提取物对外周血内皮祖细胞(endothelial progenitor cell, EPC)数量和功能的影响。方法密度梯度离心法获取外周血单个核细胞，培养7 d后，收集贴壁细胞并加入银杏叶提取物(10, 25和50 mg·L^-1)干预一定时间(6,12,24和48 h)。激光共聚焦显微镜和流式细胞仪鉴定EPC，分别观察EPC的增殖、迁移、粘附和体外血管生成能力。结果银杏叶提取物促进外周血EPC扩增，25 mg·L^-1银杏叶提取物作用24 h对EPC数量的影响最为显著(较对照组增加了1倍， p<0.01)。银杏叶提取物也显著改善了外周血EPC的粘附、迁移、增殖和体外血管生成能力。结论银杏叶提取物可增加EPC数量并改善其功能。     

熊果酸对兔外周血内皮祖细胞数量和功能的影响

目的观察熊果酸对兔外周血内皮祖细胞(EPC)数量和功能的影响并探讨其可能的作用机制。方法以密度梯度离心法分离兔外周血单个核细胞,并接种于纤连蛋白包被的培养板上,予含内皮细胞生长添加剂(ECGS)的M199培养基培养14天,应用免疫荧光染色及流式细胞仪检测、鉴定内皮祖细胞,分别以MTT法、改良的Boyden小室、黏附能力测试、ELISA法观察不同浓度(5,10,50,100mmol/L)熊果酸对培养的EPC增殖、迁移、黏附及分泌功能的影响;同时观察糖皮质激素受体拮抗剂RU486预作用的效果。结果熊果酸可浓度依赖性减少兔EPC的数量,并减弱其增殖、迁移、黏附和分泌能力;RU486可部分逆转熊果酸的上述抑制作用。结论熊果酸可能部分通过糖皮质激素受体的介导影响EPC的数量和功能,而这种抑制作用可能使其在抗动脉粥样硬化和抗再狭窄的应用中受限。     

诺帝对血管内皮生长因子诱导的人脐血源性内皮祖细胞功能的影响

探讨手性化合物诺帝(Nordy)对血管内皮生长因子(VEGF)诱导的人脐血源性内皮祖细胞(EPCs)功能的影响及其意义。应用密度梯度离心法分离新鲜人脐血的单个核细胞，接种于EGM-2培养液中培养7～10 d获得内皮祖细胞(EPCs)。分别采用MTT法、Millicell-PCF培养小室系统和Matrigel内小管形成试验检测诺帝对VEGF刺激下EPCs增殖活性、迁移能力和体外形成小管样结构能力的影响。结果表明，100 μmol·L^-1诺帝作用24 h明显抑制EPCs增殖活性(P<0.05)，诺帝(25～50 μmol·L^-1)作用48～72 h也明显抑制EPCs增殖活性(P<0.05)。诺帝(25～100 μmol·L^-1)显著抑制VEGF诱导的EPCs迁移活性和体外形成小管样结构的能力(P<0.05)。诺帝能抑制体外VEGF诱导的人脐血源性EPCs增殖、迁移和体外小管形成能力，提示其具有抗EPCs效应。     

骨化三醇对体外培养内皮祖细胞的药理作用

目的观察骨化三醇[1,25-(OH)2D3]干预培养对人外周血来源的内皮祖细胞(Endothelial progenitor cells,EPCs)数量及功能的影响。方法采用密度梯度离心法和差速贴壁法,获得EPCs细胞,用流式细胞仪和激光共聚焦显微镜进行鉴定;然后分别加入不同浓度的骨化三醇10、50、100μmol/L干预培养72 h,观察EPCs的数量变化、黏附、增殖和产生一氧化氮(NO)的能力。结果与对照组相比,10、50、100μmol/L组的贴壁细胞个数分别为13.91±1.58、14.28±1.66、16.91±1.85,差异有统计学意义(P<0.01);重贴壁细胞个数分别为9.55±1.11、10.25±1.15、10.88±1.02,差异有统计学意义(P<0.01);增殖能力(OD值)分别为0.285±0.005、0.301±0.009、0.321±0.006,差异无统计学意义(P>0.05);培养液中NO含量分别为5.687 9±0.392 5、5.954 7±0.243 2、7.682 2±0.353 4μmol/L,差异无统计学意义(P>0.05)。结论骨化三醇干预培养能提高人外周血来源EPCs的黏附能力,但对其增殖能力和产NO能力无明显影响。     

阿托伐他汀对外周血培养内皮祖细胞数量及功能的影响

目的 观察阿托伐他汀对体外培养外周血内皮祖细胞(EPCs)数量及功能的影响.方法 分离培养人外周血单个核细胞,分别接种在普通培养基(A组)及阿托伐他汀干预培养基(B组),7d后收集贴壁细胞进行分析.激光共聚焦显微镜下双染色阳性细胞鉴定为正在分化的EPCs,采用transwell小室、细胞计数法评估EPCs扩增、黏附及迁移能力的差别.结果 与A组相比,B组体外培养EPCs的数量显著增多,黏附及迁移能力显著提高.结论 阿托伐他汀在外周血EPCs培养中能促使细胞扩增,增强黏附及迁移功能,可作为EPCs培养的一种改良方法.     

血管内皮祖细胞与冠心病

<正>冠心病是病死率极高的疾病之一,其发病率仍在显著升高。通常,冠心病的治疗包括药物、介入和手术。虽然这些治疗也在不断发展,能够改善心肌缺血和心力衰竭的症状,使闭塞的血管再通,但均有其局限性。近年来的研究又发现骨髓、脐血和外周血中存在能分化为内皮细胞并参与血管新生的血管内皮祖细胞,于是在20世纪末,人们提出了用血管内皮祖细胞(EPC)治疗缺血性心脏病的构想。     

姜黄素对慢性阻塞性肺疾病患者外周血内皮祖细胞数量及功能的影响

目的观察不同浓度姜黄素对慢性阻塞性肺疾病(COPD)患者外周血内皮祖细胞(EPCs)数量、增殖功能、一氧化氮(NO)分泌功能及内皮型一氧化氮合酶(e NOS)表达的影响,探讨姜黄素修复和保护COPD患者受损的血管内皮功能作用的可能机制。方法选择30例住院的COPD患者,密度梯度离心法获取外周血单个核细胞,加入不同浓度姜黄素(0、5、10、15μmol·L-1)刺激并诱导其分化为EPCs,荧光双染色鉴定EPCs并进行细胞计数。收集贴壁细胞,再加入各浓度姜黄素,相同条件下培养一定时间(6、12、24、48 h)。采用CCK-8法检测EPCs的增殖能力,硝酸还原酶法(Griess法)检测NO分泌量,Western Blot法检测e NOS蛋白的表达。结果与未加入姜黄素的对照组相比,姜黄素可显著增加COPD患者EPCs的数量,并呈浓度依赖性;姜黄素显著改善了EPCs的增殖功能及NO分泌功能,呈浓度时间依赖性;姜黄素也可显著上调EPCs的e NOS表达,与姜黄素浓度之间具有相关性。结论姜黄素增加了EPCs的数量,改善了EPCs增殖功能及NO分泌功能,并上调了e NOS的表达。提示姜黄素可能通过增加EPCs的数量及改善EPCs的功能来修复COPD患者的血管内皮功能损伤。     

内皮祖细胞移植对缺血性血管新生的作用

广义的血管新生(neovascularization)是指产生新的血管或从已存在的血管形成毛细血管的过程.1997年Asaharad等[1]首次发现成体外周血循环中存在血管内皮祖细胞(endothelial progenitor cell,EPC),证实出生后的成体存在有血管发生(vasculogenesis)和血管生成(angiogenesis).两种生成血管机制,揭示了EPC参与机体内缺血性血管新生过程,从此拉开了EPC研究的序幕.     

丙丁酚对冠心病血管内皮功能的影响

目的观察丙丁酚（普罗布考）对冠心病血管内皮功能的影响。方法采用高分辨率超声诊断系统检测35例确诊冠心病的老年患者服用普罗布考前后肱动脉血流介导的舒张功能（即内度依赖性舒张功能，FMD）和硝酸甘油介导的舒张功能（非内皮依赖性舒张功能，NMD）的变化，同时检测治疗前后血浆一氧化氮（NO）、内皮素（ET）水平的变化，并与28例未接受调脂治疗的老年冠心病患者进行对照研究。结果接受普罗布考治疗的患者ET、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）水平明显降低，而NO和FMD明显增加，NMD无显著性改变。结论普罗布考在降脂的同时能够改善老年冠心病患者血管内皮功能，且该作用独立于高密度脂蛋白胆固醇（HDL-C）水平的降低。     

血管内皮祖细胞在恶性肿瘤血管生成中的作用

恶性肿瘤是严重影响人类健康和生命安全的疾病,新的血管生成是恶性肿瘤发生和发展的重要生理病理过程。人骨髓中含有血管内皮祖细胞（EPCs）,来源于骨髓EPCs的通过不同的机制动员进入血液循环,并参与肿瘤的血管生成。本文主要综述了EPCs的主要来源、生物学特性以及其参与肿瘤血管生成的机制。由于EPCs可以成为肿瘤诊断及治疗中的靶细胞,对其进行广泛而深入的研究将为肿瘤个体化治疗提供新策略。     

Effects of resveratrol on number and activity of endothelial progenitor cells from human peripheral blood

1. It has been well established that oestrogens can increase the number of endothelial progenitor cells (EPC) by anti-apoptotic effects. Resveratrol, a polyphenolic phytoalexin extracted from grapes and wine, has been reported to act as an oestrogen receptor agonist. We hypothesize that putative phyto-oestrogen may promote EPC proliferation and survival in vitro. 2. Endothelial progenitor cells were isolated from human peripheral blood and identified immunocytochemically. Endothelial progenitor cells were incubated with resveratrol (1, 10, 25 and 50 mmol/L) or control for specified times. Cell proliferation, migration and in vitro vasculogenesis were assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, modified Boyden chamber assay and in vitro vasculogenesis detection, respectively. 3. Resveratrol increased the number of EPC and promoted EPC proliferation, adhesion and migration in a dose- and time-dependent manner. Cell number peaked at 50 mmol/L resveratrol after incubation for 24 h compared with vehicle control (61.3 +/- 5.8 vs 112.8 +/- 7.2, respectively; P < 0.01). 4. Furthermore, cell cycle analysis showed that 50 mmol/L resveratrol significantly increased the S phase and decreased the G(0)/G(1) phase of EPC. In addition, resveratrol increased vascular endothelial growth factor production and further induced vasculogenesis in vitro. 5. In conclusion, resveratrol significantly induces EPC proliferation, migration and further promotes angiogenesis in vitro.     

Effects of puerarin on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether puerarin can augment endothelial progenitor cells (EPCs) numbers, promote EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. METHODS: EPCs were characterized as adherent cells by double staining of DiLDL-uptake and lectin binding under a laser scanning confocal microscope. Expression of KDR, VEGFR-2, and AC 133 was detected by flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis were determined with MTT assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. EPCs adhesive assay was performed by replating those on fibronectin-coated dishes, then adherent cells were counted. RESULTS: Incubation of isolated human MNCs with puerarin 0.1-3 mmol/L increased the number of EPCs, EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity in a concentration and time-dependent manner, which reached maximum at 3 mmol/L, 24 h (approximately 1-fold increase, P<0.01). CONCLUSION: Puerarin enhanced     

低分子量肝素对外周血内皮祖细胞数量和功能的影响

目的通过观察低分子量肝素(LMWH)对体外培养的外周血内皮祖细胞(EPC)数量和功能的影响,旨在探讨EPC参与LMWH治疗心血管疾病的作用机制。方法采用密度梯度离心法从外周血获取单个核细胞,培养7 d后,洗去非贴壁细胞,分别加入50,100,200和400 kU.L-1的LMWH培养一定时间(6,12,24,48和72 h)后收集细胞进行研究。激光共聚焦显微镜鉴定FITC-UEA-Ⅰ和Dil-acLDL双染色阳性细胞为正在分化的EPC,并在倒置荧光显微镜下计数。分别采用MTT比色法,改良的Boyden小室和黏附能力实验来观察EPC的增殖能力,迁移能力和黏附能力。结果LMWH能显著增加外周血EPC数量,200 kU.L-1浓度的LMWH作用48 h影响最为显著(对照vs200 kU.L-1LMWH,44±5vs112±9)。LMWH也能显著改善外周血EPC的增殖能力(对照vs200 kU.L-1LMWH,0.504±0.097vs0.828±0.109),迁移能力(对照vs200 kU.L-1LMWH,8±6vs40±8)和黏附能力(对照vs200 kU.L-1LMWH,9±4vs29±4)。结论增加EPC的数量及促进其增殖、迁移和黏附等功能是LMWH治疗心血管疾病的作用机制之一。     

Effects of Ginkgo biloba extract on number and activity of endothelial progenitor cells from peripheral blood

The aim of this study is to investigate whether Ginkgo biloba extract can augment endothelial progenitor cells numbers, and promote the cells' proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with Ginkgo biloba extract (to make a series of final concentrations: 10 mg/L, 25 mg/L, and 50 mg/L) or vehicle control for the respective time points (6 hours, 12 hours, 24 hours, and 48 h). Endothelial progenitor cells were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. Endothelial progenitor cells proliferation, migration, and in vitro vasculogenesis activity were assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. Endothelial progenitor cells adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Incubation of isolated human mononuclear cells with Ginkgo biloba extract dose- and time-dependently increased the number of endothelial progenitor cells, maximum at 25 mg/L, 24 hours (approximately 1-fold increase, P < 0.01). In addition, Ginkgo biloba extract also dose- and time-dependently promoted endothelial progenitor cells proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. The results of the present study defined a novel functional effect of Ginkgo biloba extract: the augmentation of endothelial progenitor cells with enhanced functional activity.     

Effects of aspirin on number, activity and inducible nitric oxide synthase of endothelial progenitor cells from peripheral blood

AIM: To investigate whether aspirin has an influence on endothelial progenitor cells (EPC). METHODS: Total mononuclear cells (MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation, then cells were plated on fibronectin-coated culture dishes. After 7 d of culture, attached cells were stimulated with aspirin (to achieve final concentrations of 1, 2, 5, and 10 mmol/L) for 3, 6, 12, and 24 h. EPC were characterized as adherent cells that were double positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine low density lipoprotein (DiLDL) uptake and lectin binding by direct fluorescent staining. EPC proliferation and migration were assayed using a 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and a modified Boyden chamber assay, respectively. An EPC adhesion assay was performed by replating the EPC on fibronectin-coated dishes, and then adherent cells were counted. In vitro vasculogenesis activity was assayed by using an in vitro vasculogenesis kit. Inducible nitric oxide synthase (iNOS) was assayed by Western blotting. RESULTS: Incubation of isolated human MNC with aspirin decreased the number of EPC. Aspirin also decreased the proliferative, migratory, adhesive, and in vitro vasculogenesis capacity of EPC, and also their iNOS levels in a concentration- and time-dependent manner. CONCLUSION: Aspirin decreases (1) the number of EPC; (2) the proliferative, migratory, adhesive and in vitro vasculogenesis capacities of EPC; and (3) iNOS levels in EPC.     

阿托伐他汀对慢性肾衰竭大鼠外周血内皮祖细胞的影响

目的探讨阿托伐他汀对慢性肾衰竭大鼠外周血内皮祖细胞(endothelial progenitor cells,EPCs)数量和功能的影响。方法采用分阶段5/6肾切除制备大鼠慢性肾衰竭模型。实验动物随机分为4组:假手术组、慢性肾衰竭组(模型组)、8mg.kg-1.d-1阿托伐他汀干预组(小剂量组)、16mg.kg-1.d-1阿托伐他汀干预组(大剂量组)。大鼠阿托伐他汀灌胃8周后,取其外周血分离与培养EPCs,并检测EPCs数量及其增殖、黏附能力。结果与假手术组比较,慢性肾衰竭大鼠外周血EPCs数量及其增殖、黏附能力均下降(P<0.05)。应用阿托伐他汀能明显增加慢性肾衰竭大鼠外周血EPCs数量,改善外周血EPCs增殖、黏附能力(P<0.05),并且呈剂量依赖性。结论阿托伐他汀可改善慢性肾衰竭大鼠外周血EPCs的数量和功能。     

人外周血内皮前体细胞体外增殖规律的动态观察及其特性

目的：探讨人外周血内皮前体细胞（endothelial progenitor cells EPCs）体外诱导分化为内皮细胞（ECs）的生长增殖规律及其特性，以期证实人外周血是临床治疗缺血性疾病一个理想的内皮前体细胞来源。方法密度梯度离心提取单个核细胞，接种在人纤连蛋白包被的培养板上，培养于含有促血管生长因子VEGF、b—FGF、IGF、EGF等的内皮生长基质EGM-2 MV中。每天倒置显微镜观察，并记录。4d后去除未附着细胞，继续培养黏附细胞，同时，黏附细胞用Dn—AC—LDL和FITC—UEA—1进行免疫荧光染色，荧光显微镜和共聚焦激光扫描显微镜观察。收集第7d的黏附细胞，流式细胞仪检测细胞表面标志CD34和CD31。结果接种1d后，一些细胞变形；2d后，有细胞团形成；3d后，细胞团周围一些贴壁细胞开始出现；4d后，黏附细胞呈短梭形或多角形贴壁生长，荧光显微镜、共聚焦激光扫描显微镜下可观察到Dn—AC—LDL和FITC—UEA-1双阳性的黏附细胞；第6d、7d，黏附细胞呈长梭形；FACS分析，CD34和CD31阳性率分别为（14．13±2．79）％、（54．67±3．44）％。结论外周血中确实存在EPCs，并且在促血管生长因子VEGF、b—FGF、IGF、EGF等的刺激下能分化为成熟的内皮细胞。EPCS可望作为临床血管再生的细胞治疗     

丹参酮ⅡA对外周血内皮祖细胞增殖、粘附和迁移功能的影响

丹参酮ⅡA(TanⅡA)是中药丹参(salvia miltiorrhiza Bge)中分离提取的一种有效单体。实验表明丹TanⅡA可促进心肌梗死冠脉侧支循环形成,对损伤内皮细胞功能障碍具有保护作用[1,2]。作为内皮细胞前体的内皮祖细胞(endo-thelial progenitor cell,EPC)在内皮损伤后的修复及侧支循环形成中起重要作用[3]。本文采用体外培养外周血EPC,观察TanⅡA对内皮祖细胞增殖、粘附和迁移功能的影响,以进一步探讨TanⅡA治疗冠心病的作用机制。1材料与方法1.1材料人纤维连接蛋白(HFN)和VEGF购自Chemicon公司;M199和FITC-UEA-I为Sigma产品;acLDL…     

脑钠肽对外周血内皮祖细胞增殖、迁移与黏附能力的影响

目的研究脑钠肽对外周血内皮祖细胞数量及增殖、迁移、黏附能力的影响。方法选取30例健康成人,取其外周静脉血,采用密度梯度离心法分离外周血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上,经血管内皮生长因子（Vascular endothelial growth factor,VEGF）诱导培养,7d后获得贴壁细胞,荧光显微镜鉴定FITC-UEA-I和Dil-acLDL双染色为正在分化的内皮祖细胞（endothelial progenitor cells,EPCs）。收集EPCs随机分成6组,分别为对照组和不同浓度脑钠肽干预组（0.05μg/ml、0.10μg/ml、0.15μg/ml、0.20μg/ml、0.25μg/ml）,均培养24h后采用MTT比色法测定内皮祖细胞（EPCs）的增殖能力,改良的Boyden小室法测定EPCs的迁移能力,黏附实验测定内皮祖细胞的黏附能力。结果不同浓度的重组人脑钠肽干预组均显著增加EPCs的增殖及迁移能力,分别与两者呈一定的量效关系,对黏附能力无明显影响。结论脑钠肽能促进外周血内皮祖细胞增殖、迁移能力,对内皮祖细胞的黏附能力无明显影响。