人外周血、脐血、脂肪组织来源的内皮祖细胞部分生物学特性比较

目的研究人外周血、脐血和脂肪组织三种不同来源内皮祖细胞部分生物学特性异同,探讨其可能不同的适应范围和方式及方法,进而为提高内皮祖细胞的应用效率提供依据。方法采用密度梯度离心法分离人外周血、脐血单个核细胞,胰蛋白酶消化提取脂肪组织干细胞。所采集细胞诱导分化,观察细胞形态,对培养第7天的细胞进行鉴定。采用MTT法检测细胞增殖,Boyden小室检测细胞迁移能力,贴壁法检测细胞黏附功能以及血管生成试剂盒检测细胞体外成血管能力。结果诱导分化细胞均证实为内皮祖细胞。脐血源性内皮祖细胞增殖、迁移、黏附、成血管能力最强(P<0.05)。脂肪组织来源内皮祖细胞增殖与成血管能力方面较外周血来源内皮祖细胞强(P<0.05)。结论人脐血内皮祖细胞可能更适用于同种异体干细胞移植,脂肪组织来源内皮祖细胞可能在自体干细胞移植治疗缺血性疾病方面有潜在应用前景。     

他汀类药物与内皮祖细胞

内皮祖细胞是来源于骨髓可分化为内皮细胞的一群细胞,在组织缺血及血管损伤时动员入血,参与微血管的生成及血管内皮的修复。有研究表明循环内皮祖细胞的数量和功能与冠心病危险因子呈负相关,提示循环内皮祖细胞有作为心血管疾病预后评估工具的应用前景。他汀类药物除了调节血脂的作用外,在血脂正常的动物模型中也发现能增加内皮祖细胞数量及增强内皮祖细胞功能,他汀类药物的这种作用可能是其临床上治疗冠心病获益的机制之一。     

自体内皮祖细胞移植修复兔动脉内皮损伤

目的观察不同数量的内皮祖细胞移植对内膜球囊损伤血管修复的影响。方法密度梯度离心法分离兔外周血单个核细胞,以EGM-2培养基培养7天,获得兔外周血内皮祖细胞。分高细胞数量移植组(5×105个细胞)和低细胞数量移植组(2×105个细胞),将相应数量的内皮祖细胞重悬于100μL生理盐水中移植至兔内膜球囊损伤血管局部,对照组仅注入100μL生理盐水。同时用CM-DiI标记内皮祖细胞移植,进行细胞示踪。4周后,处死动物,荧光显微镜下观察CM-DiI标记细胞的分布,并测量各组内膜损伤血管再生内皮覆盖率和新生内膜增生程度。结果荧光显微镜下发现荧光标记的内皮祖细胞分布在血管的中膜层、新生内膜层和内膜表面。内皮祖细胞移植可显著促进内膜损伤血管的再内皮化,以高细胞数量移植组为著,同时内皮祖细胞移植也大大减少了新生内膜的形成。结论内皮祖细胞移植可修复内膜球囊损伤血管,高细胞数量移植有更显著的效果。     

他汀类药物对血管新生作用及机制的研究

小剂量他汀类药物能促进骨髓来源内皮祖细胞的动员和分化,提高内皮祖细胞的数量和分化、迁移能力,促进缺血组织血管新生。小剂量他汀类药物可能通过磷脂酰肌醇-3激酶(PI-3K)／苏氨酸激酶(Akt)旁路增加内皮型一氧化氮合酶的表达,从而动员内皮祖细胞。而大剂量他汀类药物可抑制血管新生。     

冠心病患者内皮祖细胞变化与冠状动脉病变的相关性

目的观察冠心病患者外周血中提取的内皮祖细胞的细胞形态、数量、集落数与正常对照组的区别。并研究冠心病患者冠状动脉狭窄的不同范围和程度与内皮祖细胞数量变化的相关性。方法选择冠心病患者57例和对照组30例,从外周血获取单个核细胞,体外培养后进行细胞分析和计数。并分析冠状动脉狭窄的不同范围和程度,患者内皮祖细胞数量和成集落数量的区别,并进行相关分析。结果 (1)冠心病患者外周血内皮祖细胞数量(23.1±1.8比56.7±2.4)和细胞集落数(14.7±2.5比24.2±1.7)较对照组明显减少;(2)随着冠状动脉狭窄范围的扩大和狭窄程度的加重,内皮祖细胞的数量和活性明显下降。结论冠心病患者外周血内皮祖细胞的数量和成集落的数量明显降低;冠心病患者外周血内皮祖细胞的数量与冠状动脉病变的范围和狭窄程度呈负相关。     

他汀类药物对血管新生作用及机制的研究

小剂量他汀类药物能促进骨髓来源内皮祖细胞的动员和分化,提高内皮祖细胞的数量和分化、迁移能力,促进缺血组织血管新生.小剂量他汀类药物可能通过磷脂酰肌醇-3激酶(PI-3K)/苏氨酸激酶(Akt)旁路增加内皮型一氧化氮合酶的表达,从而动员内皮祖细胞.而大剂量他汀类药物可抑制血管新生.     

大鼠血管内皮祖细胞培养及鉴定

目的探索骨髓来源的血管内皮祖细胞的分离培养方法,为缺血性疾病治疗找到新的移植细胞来源。方法采集SD雄性大鼠骨髓细胞,用梯度密度离心法分离单核个细胞,种植于提前包埋了纤维连接蛋白的培养皿中培养,将血管内皮生长因子(VEGF)、重组纤维细胞生长因子(bFGF)和重组上皮生长因子(EGF)作为诱导剂,将72h后贴壁细胞(A组)和非贴壁细胞(B组)分别培养,观察细胞形态的变化以及数量变化,取A组在第14天,应用免疫组化和免疫荧光技术鉴定内皮祖细胞系列标志。结果A组细胞经过体外诱导后大量扩增,在28d时仍有强大扩增的活力,呈铺路石样,而B组细胞短期内可以大量增殖,呈长索样,之后逐渐变少;在细胞迁移实验中,A组细胞可以相互融合形成血管结构,而B组细胞之间则不能融合,随着时间推移,逐渐凋亡;A组经过CD34免疫荧光、CD133、FLK-1免疫组化鉴定呈阳性,并能特异性吸附FITC-UEA-和内吞DIL-Ac-LDL。结论自体骨髓细胞通过贴壁换液后可以分离培养出内皮祖细胞,取贴壁细胞经过体外诱导后大量扩增,并通过表面标记物鉴定可以确认为内皮祖细胞,而非贴壁细胞不能大量增殖,故将贴壁分离方法来筛选内皮祖细胞,此方法简易,并能满足细胞移植的需要,因而,在移植治疗脑缺血引起的内皮损伤应该有较好的临床应用前景。     

烟雾病治疗中外周血内皮祖细胞及其生物学特性的研究

目的探讨烟雾病患者在接受保守治疗和手术治疗前后外周血中内皮祖细胞(endothelial progenitor cells, EPCs)的变化情况。方法选取2015年6月至2017年6月在沧州市中心医院接受治疗的烟雾病患者60例作为研究对象。其中接受药物保守治疗患者30例为保守治疗组,接受颅内外脑血管搭桥手术治疗患者30例为手术治疗组。所有患者均于治疗前和治疗后6个月留取外周血。将外周血经密度梯度离心法分离出单个核细胞,利用流式细胞仪检测外周血中内皮祖细胞数量。分别检测两组内皮祖细胞的增殖和迁移能力。结果手术治疗组治疗6个月后的内皮祖细胞数量较治疗前降低(t=5.761,P 0.05),手术治疗组较保守治疗组治疗后6个月的内皮祖细胞数量更低(t=4.202,P0.05)。保守治疗组和手术治疗组内皮祖细胞治疗前后的增值能力变化差异无统计学意义(t=0.540、0.820,P 0.05),两组迁移能力变化也无统计学意义(t=0.900、-0.361,P 0.05)。结论手术治疗较保守治疗能降低烟雾病患者外周血中内皮祖细胞的数量,但对于内皮祖细胞增殖和迁移能力无明显影响。     

干细胞/组细胞与动脉粥样硬化研究进展

动脉粥样硬化及其相关并发症是人类死亡的主要原因。与其他危险因素相比,越来越多学者认为衰老是动脉粥样硬化的主要危险因素。60岁以上,年龄将支配风险预测中所有其他风险因素。然而老龄化风险的具体机制尚不清楚。近年来,干细胞/祖细胞与疾病的相关性研究突飞猛进,血管祖细胞与血管性疾病的关系日益受到人们的关注。1997年Asahara首次报道了在循环血液中有争议的内皮祖细胞的分离。这些细胞能够在体外分化为成熟的内皮细胞,并结合于血管生成的活性位点。这一发现开创了血管生物学的新时代,也使研究者们对动脉粥样硬化及血栓栓塞等并发症开始重新认识。提出了动脉粥样硬化风险的一个新的概念:在动脉粥样硬化的发展过程中,存在修复损伤内皮的内皮祖细胞耗竭,即内皮祖细胞依赖性的动脉修复缺乏。平滑肌祖细胞则是斑块内平滑肌源性泡沫细胞的重要来源之一,参与了斑块的形成。本文重点阐述动脉粥样硬化发生发展中干细胞/组细胞的作用。尤其是内皮祖细胞、平滑肌祖细胞与动脉粥样硬化的关系及血管祖细胞的分化等问题。     

未来治疗的新领域——内皮祖细胞

内皮祖细胞来源于骨髓,可分化为成熟有功能的内皮细胞.大量试验证实冠心病和心力衰竭患者内皮祖细胞数量减低,外周循环内皮祖细胞功能受损,许多因素可影响内皮祖细胞的数量及功能.因此,药物动员内皮祖细胞或使其功能正常化有望改善心血管病患者的预后.现总结近来关于影响内皮祖细胞因素的研究,有助于探索未来治疗的新领域.     

Role of progenitor endothelial cells in cardiovascular disease and upcoming therapies.

The field of cell-based transplantation has expanded considerably and is poised to become an established cardiovascular therapy in the near future. In this review, we will focus on endothelial progenitor cells (EPCs), which are immature cells capable of differentiating into mature endothelial cells. EPCs share many surface marker antigens such as CD34, AC133, Flk-1, etc. with hematopoietic stem cells (HSCs) and the major source of EPCs as well as HSCs is the bone marrow (BM). BM-derived EPCs are mobilized into peripheral blood and recruited to the foci of pathophysiological neovascularization and reendothelialization, thereby contributing to vascular regeneration. Severe EPC dysfunction is an indicator of poor prognosis and severe endothelial dysfunction. Indeed, number of circulating EPCs and their migratory activity are reduced in patients with diabetes, coronary artery disease (CAD), or subjects with multiple coronary risk factors. Effective neovascularization induced by EPC transplantation for hindlimb, myocardial, and cerebral ischemia has been demonstrated in many preclinical studies, and early clinical trials of EPC transplantation in chronic and acute CAD indicate safety and feasibility of myocardial cell-based therapies. For therapeutic reendothelialization in patients undergoing percutaneous coronary intervention, CD34 antibody-coated stents have been used clinically to capture circulating EPCs at the injury sites and enhance reendothelialization and safety of stents. Further development in cell processing technology for efficient isolation, expansion, mobilization, recruitment, and transplantation of EPCs into target tissues are underway and expected to be tested in clinical trials in the near future.     

氯沙坦对冠心病外周血内皮祖细胞的动员作用及影响内皮功能的机制

目的观察氯沙坦对冠心病患者外周血内皮祖细胞(EPCs)数量及血流介导的内皮舒张功能的影响,并探讨其改善内皮功能的机制。方法选取经冠脉造影确诊为冠心病65例,随机分为标准治疗组32例和ARB治疗组33例,同时选取非冠心病31例作为健康对照组。标准治疗组给予常规药物,ARB治疗组给予常规物药+氯沙坦治疗,随访8周,治疗前后分别用流式细胞术检测EPCs水平、超声测定流量介导血管扩张(FMD)情况。结果基线水平,冠心病内皮功能较健康对照组明显低下、EPCs数量明显减少(P<0.05);FMD与EPCs数量呈正相关(r=0.57,P<0.01)。治疗8周后,标准治疗组和ARB治疗组冠心病患者FMD均有不同程度的改善,循环血EPCs数量均有不同程度的增加,但是ARB治疗组变化更明显(P<0.05);ARB治疗组EPCs的增加与内皮功能的改善呈正相关(r=0.52,P<0.01)。结论氯沙坦对EPCs具有一定的动员作用,ARB类药物氯沙坦可能通过增加外周血EPCs数量,进而改善内皮功能。     

雌二醇对骨髓内皮祖细胞部分生物学功能的影响

目的观察雌二醇对体外培养骨髓来源内皮祖细胞(endothelial progenitor cells,EPCs)数量及增殖、迁移、黏附功能的影响。方法密度梯度离心法获取骨髓单个核细胞,FITC-荆豆凝集素I、DiI-乙酰化低密度脂蛋白荧光双染鉴定。单个核细胞培养4天后进行实验分组,分为对照组和雌二醇治疗组。雌二醇治疗组加入不同浓度雌二醇(分别为0.001、0.01、0.1μmol/L)培养48 h,然后分别采用四氮唑溴盐比色法、改良的Boyden小室和黏附能力测定来观察EPCs的增殖、迁移和黏附能力。结果雌二醇剂量依赖性增加EPCs数量并显著改善了EPCs的黏附、迁移和增殖能力,与对照组比较差异显著。结论雌二醇可增加培养EPCs的数量并改善EPCs部分生物学功能。     

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.     

辛伐他汀对冠心病患者内皮祖细胞增殖的影响及其机制初步探讨

目的:观察辛伐他汀对冠心病患者外周血内皮祖细胞(EPCs)增殖的影响并初步探讨其机制。方法:冠心病患者28例,随机分为辛伐他汀组和对照组,前者给予辛伐他汀40mg/d口服,治疗前及治疗后2、4周抽取外周血,采用密度梯度离心法获取单个核细胞,培养7d后对贴壁的细胞进行分析。以激光共聚焦显微镜鉴定为FITC标记的荆豆凝集素Ⅰ和Dil(一种亲脂性碳化青荧光染料)标记的乙酰化低密度脂蛋白双染色阳性细胞为EPCs。计数法比较二组EPCs数目,并对病人血浆低密度脂蛋白-胆固醇(LDL-C)水平和EPCs数进行相关分析。另取10例冠心病患者血培养EPCs,加入辛伐他汀、PI3K/Akt信号转导通路抑制剂Wortmannin后观察对其增殖的影响。结果:辛伐他汀组病人治疗后2周、4周外周血中EPCs明显上升(P<0.01),其变化与LDL-C变化无相关性(P>0.05),体外实验辛伐他汀 Wortmannin组的血EPC数目较辛伐他汀组明显下降(P<0.01)。结论:辛伐他汀能刺激EPCs的增殖,这种作用能被Wortmannin阻断。故辛伐他汀作用可能与激活PI3K/Akt信号转导通路有关。     

Synergistic effect of combined intramyocardial CD34+ cells and VEGF2 gene therapy after MI

Previous studies have shown that local angiogenic gene therapy acts, in part, by recruiting endothelial progenitor cells (EPCs) to ischemic tissue. Recent data indicate that patients with the most severe vascular disease may have insufficient or deficient EPCs and the poorest response to angiogenic therapy. Accordingly, we hypothesized that combining human CD34(+) cell implantation with local vascular endothelial growth factor 2 (phVEGF2) gene therapy might overcome these deficiencies. The addition of VEGF2 to EPC cultures resulted in significant and dose-dependent decreases in EPC apoptosis. Phosphorylated Akt (p-Akt) was increased in VEGF2-treated EPCs. In vivo, myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in 34 immunodeficient rats. The animals were then randomized to one of four treatment groups: cell therapy alone with human CD34(+) cells; VEGF2 gene therapy alone; combination therapy with CD34(+) cells plus phVEGF2; or CD34(-) cells and 50 microg empty plasmid. Four weeks after MI, animals treated with combination therapy showed improved fractional shortening, increased capillary density, and reduced infarct size compared with the other three groups. Combination therapy was also associated with an increased number of circulating EPCs 1 week after MI. Combined subtherapeutic doses of cell and gene therapy result in a significant therapeutic effect compared to monotherapy. This approach may overcome therapeutic failures (e.g. inability of certain patients to mobilize sufficient EPCs) and may also offer safety advantages by allowing lower dosing strategies.     

SDF-1对小鼠骨髓源性内皮祖细胞数量及功能的影响

目的:观察基质细胞衍生因子-1(SDF-1)对体外培养的小鼠骨髓源性内皮祖细胞(EPC s)数量及功能的影响。方法:以密度梯度离心法获取小鼠骨髓单个核细胞(M NC s),由BS-1和D iI-acLDL荧光双染鉴定。加入不同浓度SDF-1 a培养48 h,然后分别采用M TT法、改良的Boyden小室和粘附能力测定来观察EPC s的增殖、迁移和粘附能力;采用血清饥饿法和紫杉醇诱导EPC s凋亡,TUNEL法和流式细胞仪检测SDF-1a对EPC s凋亡率的影响。结果:SDF-1a剂量依赖性增加EPC s数量(P<0.01),并显著改善了EPC s的粘附、迁移和增殖能力(P<0.01),显著降低EPC s凋亡率(P<0.01)。结论:SDF-1a可增加小鼠骨髓源性EPC s的数量并改善EPC s功能。     

高胆固醇对内皮祖细胞数量和功能的影响

目的　观察高胆固醇血症患者外周血内皮祖细胞 (EPC)数量和功能的变化。方法选择高胆固醇血症患者和非高胆固醇血症患者各 2 0例 ,用密度梯度离心法从外周血获取单个核细胞 ,将其接种在人纤维连接蛋白包被培养板 ,培养 7d后贴壁细胞进行细胞化学分析。激光共聚焦显微镜鉴定FITC UEA Ⅰ和DiI acLDL双染色阳性细胞为正在分化的EPC。采用MTT比色法、改良的Boyden小室和黏附能力测定实验观察EPC的增殖、迁移和黏附能力。结果　高胆固醇血症患者外周血EPC数量明显减少 [(41 8± 8 7比 6 4 5± 16 6 )EPCs/× 2 0 0视野 ,P <0 0 5 ],且EPC数量与血总胆固醇水平 (r =- 0 6 5 9,P <0 0 0 1)和低密度脂蛋白胆固醇水平 (r =- 0 6 11,P <0 0 0 1)呈反向线性关系。高胆固醇血症患者EPC的增殖、迁移和黏附能力也明显受损。结论　高胆固醇患者外周血内皮祖细胞数量减少、功能减退。     

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice

AimDysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D).MethodsThe colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKA^y mice, using EPC colony-forming assay (EPC-CFA).ResultsT2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types.ConclusionT2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.     

The number of endothelial progenitor cell colonies in the blood is increased in patients with angiographically significant coronary artery disease.

OBJECTIVES: The objective of this study was to determine whether the number of endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs) in peripheral blood was associated with the presence and severity of coronary artery disease (CAD) in patients undergoing coronary angiography. BACKGROUND: Previous studies have suggested an inverse relationship between levels of circulating EPCs/CACs and the presence of CAD or cardiovascular risk factors, whereas other studies have observed increased numbers of EPCs in the setting of acute ischemia. However, the criteria used to identify specific angiogenic cell subpopulations and methods of evaluating CAD varied in these studies. In the present study, we used rigorous criteria to identify EPCs and CACs in the blood of patients undergoing coronary angiography. METHODS: The number of EPCs and CACs were measured in the blood of 48 patients undergoing coronary angiography. Patients with acute coronary syndromes were excluded. RESULTS: Compared with patients without angiographically significant CAD, the number of EPCs was increased (1.11 +/- 2.50 vs. 4.01 +/- 3.70 colonies/well, p = 0.004) and the number of CACs trended higher (175 +/- 137 vs. 250 +/- 160 cells per mm(2), p = 0.09) among patients with significant CAD. The highest levels of EPCs were isolated from patients subsequently selected for revascularization (5.03 +/- 4.10 colonies/well). CONCLUSIONS: In patients referred for coronary angiography, higher numbers of EPCs, and a trend toward higher numbers of CACs, were associated with the presence of significant CAD, and EPC number correlated with maximum angiographic stenosis severity. Endothelial progenitor cell levels were highest in patients with CAD selected for revascularization.