Increased levels of circulating endothelial progenitor cells and circulating endothelial nitric oxide synthase in patients with gliomas

OBJECTIVE: Gliomas are among the highest vascularized tumors. We hypothesized that patients with gliomas have increased levels of circulating endothelial progenitor cells (EPCs) and circulating endothelial nitric oxide synthase (eNOS). METHODS: The fraction of EPCs was quantified by fluorescence-activated cell sorter analysis using anti-CD34, -CD133 and -KDR (kinase insert domain receptor) monoclonal antibodies in unselected peripheral blood samples of 32 patients with gliomas. Control groups included 47 patients with other central nervous system tumors or diseases, 10 patients with recent ischemic strokes, and 19 healthy blood donors. The circulating eNOS concentration of plasma was measured by a colorimetric assay in the same samples. In addition, CD34(+)CD105(+) KDR(+) and CD34(+)CD146(+)KDR(-) cell fractions were measured. RESULTS: The percentage of CD34(+)CD133(+)KDR(+) EPCs in the blood of glioma patients is significantly greater than that in the blood of patients with other central nervous system tumors or diseases (p = 0.003), stroke patients (p = 0.005), or healthy donors (p = 0.013). The plasma eNOS concentration is also significantly greater in glioma patients compared with each of the control groups (p < 0.001 for all groupwise comparisons). No significant differences in the levels of the EPCs or eNOS between any of the control groups were demonstrated. In the glioma patients, the level of eNOS correlated with the fraction of CD34(+)CD105(+)KDR(+) cells (r = 0.748; p = 0.008). INTERPRETATION: The data are suggestive of increased mobilization of EPCs contributing to neoplastic vasculogenesis in glioma. The increased levels of EPCs and eNOS in the peripheral blood of glioma patients trigger further investigations as to their value as independent parameters for use in clinical practice.     

大鼠骨髓间充质干细胞和内皮祖细胞的分离培养及鉴定

背景：骨髓间充质干细胞的分离和培养扩增至今缺乏统一的方法。内皮祖细胞可以由外周血或骨髓中的单个核细胞诱导分化而成,但因获取骨髓单个核细胞的方法各异,分离效果亦有较大差异。 目的：拟在体外分离培养大鼠骨髓间充质干细胞和内皮祖细胞,并对其进行鉴定。 设计、时间及地点：细胞学体外观察,于2008-10/2009-02在安徽医科大学第一附属医院中心实验室完成。 材料：清洁级健康雄性SD大鼠2只,由安徽省动物中心提供。 方法：抽取大鼠骨髓,通过密度梯度离心法提取单个核细胞,采用差速贴壁法分离骨髓间充质干细胞。原代骨髓间充质干细胞培养48 h后,收集未贴壁细胞,加入含胎牛血清、血管内皮生长因子、成纤维细胞生长因子、胰岛素样生长因子1、重组人上皮生长因子的EGM-2完全培养液诱导骨髓间充质干细胞向内皮祖细胞分化。设立3组：分别为早期贴壁且普通培养的内皮祖细胞、2次贴壁且诱导培养的内皮祖细胞、大鼠成熟主动脉内皮细胞,采用硝酸还原酶法间接测量细胞培养液中一氧化氮的含量。 主要观察指标：骨髓间充质干细胞与内皮祖细胞体外培养情况,细胞培养液中一氧化氮的含量。 结果：原代培养的骨髓间充质干细胞呈梭形,24 h内大部分细胞贴壁,9~10 d可达90%融合,纯化扩增后骨髓间充质干细胞呈均匀一致的长梭性,传代周期为8 d左右,流式细胞仪检测第3代骨髓间充质干细胞CD34,CD45呈阴性,CD105呈阳性。2次贴壁的内皮祖细胞培养3 d内贴壁,6 d形成集落,8~10 d出现条索状结构,呈微血管样生长,2周时大部分细胞呈多角形,细胞集落相互连接,呈典型的“铺路石”样,7~10 d细胞DiI-acLDL、FITC-UEA-1双染呈阳性,流式细胞仪检测Flk-1,CD133阳性。2次贴壁且诱导培养的内皮祖细胞培养液中一氧化氮含量明显高于早期贴壁且普通培养的内皮祖细胞(P < 0.05),但低于大鼠成熟主动脉内皮细胞(P < 0.05)。 结论：差速贴壁法能有效地分离、纯化、扩增大鼠骨髓间充质干细胞,加入多种细胞生长因子诱导后具有较强地向内皮细胞方向分化的能力,且2次贴壁细胞已具有部分内皮细胞的功能。     

Detection and quantification of mature circulating endothelial cells using flow cytometry and immunomagnetic beads: a methodological comparison

Mature circulating endothelial cells (CECs) are novel cellular markers of endothelial damage/dysfunction. The two main techniques of CEC enumeration are flow cytometry (FC) and immunomagnetic bead (IB) isolation. Both quantify CECs accurately, but a direct comparison of both methods has not been reported. We sought to assess the agreement between the two methods in two patient populations, and a group of healthy subjects, with emphasis given to methodological issues. We included 34 patients with acute coronary syndrome (ACS), 60 patients with primary breast cancer (PBC) and 30 healthy controls (HC). We quantified CECs using the IB method [CD146 and FITCUlex europaeus lectin-1] and FC [CD45, CD34 and CD146]. Bland-Altman plots suggested reasonable agreement (<5% of events >2 standard deviations from the mean) between FC and the IB methods for CEC quantification in whole blood in the two disease groups (ACS and PBC), but not among the HCs. There were no statistically significant differences in CEC levels by the two methods amongst all three patient groups. There is reasonable agreement between the FC and the IB methods for mature CEC quantification in whole blood, especially amongst disease groups. The agreement between the two methods appears to weaken in healthy controls, and at lower and higher absolute CEC counts.     

Circulating endothelial cells in pulmonary hypertension

The pulmonary endothelium plays a significant role in the pathobiology of Primary Pulmonary Hypertension. A number of diseases, related by a history of vascular injury, are associated with increased numbers of circulating endothelial cells (CECs). We hypothesized that patients with pulmonary hypertension would also have an increased number of circulating endothelial cells due to the high pressures and increased shear stress present within the pulmonary vasculature. We isolated the CECs from 14 patients with pulmonary hypertension, (5 primary and 11 secondary) and compared them to the cells from 12 normal controls. There was a significant increase in the number of CECs in peripheral blood in patients with both PPH and secondary pulmonary hypertension (SPH) when compared to normal volunteers (33.1 +/- 1.9 [PPH] and 27.2 +/- 6.9 [SPH] vs. 3.5 +/- 1.3 [controls], p < 0.001). The number of circulating endothelial cells in the patient's peripheral blood correlated significantly with the systolic, diastolic and mean pulmonary artery pressures of the individual. Approximately 50% of the CECs from patients with pulmonary hypertension expressed CD36, a marker of microvascular origin and 25% expressed E-selectin, a marker of endothelial cell activation. Although the origin of the CECs in patients with PH requires further investigation, one possible source is the pulmonary vasculature, and in patients with plexogenic pulmonary hypertension, the plexiform lesions. CECs may provide a non-invasive mean of accessing cells important to the pathobiology of severe pulmonary hypertension.     

Circulating endothelial progenitor cells and depression: a possible novel link between heart and soul

Although depression is known to be an independent risk factor for cardiovascular disorders, the mechanisms behind this connection are not well understood. However, the reduction in the number of endothelial progenitor cells (EPCs) in patients with cardiovascular risk factors has led us to hypothesize that depression influences the number of EPCs. EPCs labeled with CD34, CD133 and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood (PB) of 33 patients with a current episode of major depression and of 16 control subjects. Mature (CD34+/VEGFR2+) and immature (CD133+/VEGFR2+) EPC counts were decreased in patients (vs controls; P<0.01 for both comparisons), and there was a significant inverse relationship between EPC levels and the severity of depressive symptoms (P<0.01 for both EPC phenotypes). Additionally, we assayed the plasma levels of VEGF, C-reactive protein (CRP) and tumor necrosis factor (TNF)-alpha and observed significantly elevated TNF-alpha concentrations in patients (vs controls; P<0.05) and, moreover, a significant inverse correlation between TNF-alpha and EPC levels (P<0.05). Moreover, by means of a quantitative RT-PCR approach, we measured CD34, CD133 and VEGFR2 mRNA levels of PB samples and found a net trend toward a decrease in all the investigated EPC-specific mRNA levels in patients as compared with controls. However, statistical significance was reached only for VEGFR2 and CD133 levels (P<0.01 for both markers). This is the first paper that demonstrates evidence of decreased numbers of circulating EPCs in patients with a current episode of major depression.     

促红细胞生成素对大鼠内皮祖细胞生物学特点的影响

背景：内皮祖细胞是内皮细胞的前体细胞,可参与新生血管的形成。研究发现,促红细胞生成素对骨髓内皮祖细胞有动员作用,促进血管的新生和损伤组织修复。 目的：观察不同浓度促红细胞生成素对体外培养的大鼠内皮祖细胞功能的影响。 方法：提取Wistar大鼠股骨和胫骨骨髓,利用密度梯度离心方法分离单核细胞进行培养,分别在培养基中加入0,4,8 U/mL不同浓度的促红细胞生成素进行培养。培养7 d后,对内皮祖细胞利用FITC-UEA-1和 Dil-Ac-LDL共同染色方法进行鉴定;同时利用CD34和CD133共同标记,以流式细胞仪进行鉴定;采用黏附实验、迁移实验及MTT实验对其功能进行评定。 结果与结论：培养到7 d左右,细胞形态变化完全,细胞之间形成联接,并可以围成似管腔状形态,十二三天时,细胞融合,形成铺路石样。培养细胞荧光染色显示内皮祖细胞呈双阳性染色,经流式细胞仪鉴定培养的内皮祖细胞可以结合CD133和CD34。黏附实验、迁移实验及MTT实验显示,促红细胞生成素呈剂量依赖性提高内皮祖细胞的黏附、迁移和增殖能力。说明在0~8 U/mL浓度内,促红细胞生成素呈剂量依赖性提高内皮祖细胞的黏附、迁移和增殖功能。     

Circulating endothelial cells in acute ischaemic stroke

Increased numbers of CD146-bearing circulating endothelial cells (CECs) in the peripheral blood probably represent the most direct evidence of endothelial cell damage. As acute ischaemic strokes are associated with endothelial abnormalities, we hypothesised that these CECs are raised in acute stroke, and that they would correlate with the other indices of endothelial perturbation, i.e. plasma von Willebrand factor (vWf) and soluble E-selectin. We studied 29 hypertensive patients (19 male; mean age 63 years) who presented with an acute stroke and compared them with 30 high risk hypertensive patients (21 male; mean age 62 years) and 30 normotensive controls (16 male; mean age 58 years). CECs were estimated by CD146 immunobead capture, vWf and soluble E-selectin by ELISA. Patients with an acute ischaemic stroke had significantly higher numbers of CECs/ml of blood (p<0.001) plasma vWf (p=0.008) soluble E-selectin (p=0.002) and higher systolic blood pressure (SBP) as compared to the other groups. The number of CECs significantly correlated with soluble E-selectin (r=0.432, p<0.001) and vWf (r=0.349, p=0.001) but not with SBP (r=0.198, p=0.069). However, in multivariate analysis, only disease group (i.e. health, hypertension or stroke) was associated with increased CECs. Acute ischaemic stroke is associated with increased numbers of CECs. The latter correlate well with established plasma markers of endothelial dysfunction or damage, thus unequivocally confirming severe vasculopathy in this condition. However, the greatest influence on CECs numbers was clinical group.     

Circulating endothelial cells. Biomarker of vascular disease

Recent research has recognised new populations of non-hematopoietic cells in the blood. One of these, circulating endothelial cells (CECs), often defined by the expression of membrane glycoprotein CD146, are rarely found in the blood in health, but raised numbers are present in a wide variety of human conditions, including inflammatory, immune, infectious, neoplastic and cardiovascular disease, and seem likely to be evidence of profound vascular insult. An additional population are endothelial progenitor cells, defined by the co-expression of endothelial and immaturity cell surface molecules and also by the ability to form colonies in vitro. Although increased numbers of CECs correlate with other markers of vascular disease, questions remain regarding the precise definition, cell biology and origin of CECs. For example, they may be damaged, necrotic or apopototic, or alive, and could possess procoagulant and/or proinflammatory properties. However, since these cells seem to be representative of in situ endothelium, their phenotype may provide useful information. Indeed, whatever their phenotype, there is growing evidence that CECs may well be a novel biomarker, the measurement of which will have utility in various clinical settings related to vascular injury. Despite this promise, progress is impeded by the diversity of methodologies used to detect these cells. Accordingly, results are sometimes inconclusive and even conflicting. Nevertheless, increased CECs predict adverse cardiovascular events in acute coronary syndromes, suggesting they may move from being simply a research index to having a role in the clinic. The objective of the present communication is to condense existing data on CECs, briefly compare them with progenitor cells, and summarise possible mechanism(s) by which they may contribute to vascular pathology.     

Decreased level and defective function of circulating endothelial progenitor cells in children with moyamoya disease

Circulating endothelial progenitor cells (EPCs) play an important role in physiological and pathological neovascularization and may be involved in attenuating ischemic diseases. This study aimed to characterize circulating EPCs in moyamoya disease (MMD), one of the most common pediatric cerebrovascular diseases. Twenty‐eight children with MMD prior to any surgical treatment and 12 healthy volunteers were recruited. Peripheral blood mononuclear cells (PBMNCs) were isolated and cultured in endothelial cell growth medium. Temporal change of phenotype of cells was analyzed on days 0 and 7. The formation of EPC clusters was evaluated on day 7. The CD34⁺, CD133⁺, and KDR⁺ cells, and the number of EPC clusters was significantly reduced in children with MMD. In controls, CD34⁺ cells were significantly decreased on day 7 compared with day 0, but in MMD they were only slightly decreased. The change in KDR⁺ cells on day 7 compared with day 0 was the reverse of that for CD34⁺ cells. Functional assay of EPC demonstrated less tube formation and increased senescent‐like phenotype in children with MMD. Analysis of the circulating EPCs of MMD children reveals decreased level and defective function. This study suggests that circulating EPCs may be associated with MMD pathogenesis. © 2009 Wiley‐Liss, Inc.     

The effect of aspirin on endothelial progenitor cell biology: Preliminary investigation of novel properties

Atherosclerosis develops in an environment of endothelial injury and inflammation. Circulating endothelial progenitor cells (EPCs) are required for vascular repair and restoration of normal endothelial function. We tested the hypothesis that the nonselective cyclooxygenase (COX) inhibitor aspirin (ASA) exerts an effect on circulating EPCs.

Methods

As part of a larger study evaluating the effect of aspirin dose in primary and secondary prevention, subjects (n = 32) were assigned randomly to either 81 mg or 325 mg aspirin daily for two months, and circulating mononuclear cells were enumerated at the beginning of the study and after 2 months using fluorescent antibodies against CD34 and CD133 as well as based on aldehyde dehydrogenase (ALDH) activity. Brachial artery endothelial function via flow-mediated dilation (BAFMD) and light transmittance platelet aggregometry in response to physiologic agonists was also determined.

Results

Subjects taking aspirin at the time of study entry had a lower numbers of CD133+/34+ cells compared to those not previously exposed (0.01% vs. 0.05% of MNCs, P < 0.03). After 2 months, subjects randomized to 81 vs. 325 mg of ASA had no significant differences in the median numbers of EPCs, although mean numbers trended lower in the high dose group. Patients on chronic ASA therapy continued to have lower numbers of EPCs. Similar effects were observed in CD34 and CD 133 single-positive cells, as well as ALDH^br cells. BAFMD did not differ nor change significantly over time between aspirin dose groups. All patients had decreased ex vivo platelet aggregation in response to arachidonic acid and ADP stimulation.

Conclusions

Our preliminary studies suggest that aspirin exerts a time-dependent effect on circulating EPCs. Short-term exposure to differing doses of ASA had indeterminate effects on EPCs levels, suggesting that time of ASA exposure may play a more important role than dose. Determining the responsible mechanism(s) and the overall clinical relevance of these findings will require further investigation.     

未破裂颅内动脉瘤患者外周血内皮祖细胞数量的研究

目的 研究颅内动脉瘤与外周血中内皮祖细胞(EPCs)数量的关系.方法 选择未破裂颅内动脉瘤患者24例和正常对照者16例,用密度梯度离心法获取外周血单个核细胞,加入培养基进行选择性培养,分析14 d后细胞形成的集落数,并对贴壁细胞进行细胞鉴定、分析和计数.激光共聚焦显微镜下观察其吞噬荆豆凝集素和乙酰化低密度脂蛋白的内皮功能;用流式细胞仪检测CD34、CD133和血管内皮生长因子受体2(VEGFR-2)三抗阳性细胞并计数.结果 颅内动脉瘤患者外周血EPCs计数较对照组明显减少,流式分析结果显示三抗阳性细胞数比例有显著性差异.结论 颅内动脉瘤患者外周血EPCs的数量较无动脉瘤者明显降低.     

Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage

Breakdown of blood-brain barrier,formed mainly by brain microvascular endothelial cells(BMECs),represents the major cause of mortality during early phases of ischemic strokes.Hence,discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine.Although endothelial progenitor cells(EPCs)represent one such agents,their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells(OECs)as a result.Functional analyses of these cells,in the present study,demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel,prominent endothelial cell and angiogenesis markers,respectively.Further analyses by flow cytometry demonstrated that OECs expressed specific markers for sternness(CD34),immaturity(CD133)and endothelial cells(CD31)but not for hematopoietic cells(CD45).Like BMECs,OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes,suggesting their capacity to form tight junctions.Ischemic injury mimicked by concurrent deprivation of oxygen and glucose(4 hours)or deprivation of oxygen and glucose followed by reperfusion(20 hours)affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux,respectively.Wound scratch assays comparing the vasculoreparative capacity of cells revealed that,compared to BMECs,OECs possessed a greater proliferative and directional migratory capacity.In a triple culture model of BBB established with astrocytes,pericytes and BMEC,exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions.Taken together,these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain(neuro)vascular homeostasis during or after a cerebral ischemic injury.     

Difficulties in work-related activities among migraineurs are scarcely collected: results from a literature review

Circulating endothelial progenitor cells (EPCs) play a critical role in maintaining endothelial integrity and keeping vascular homeostasis. Previously, we reported that EPCs were involved in repair and remodeling of aneurismal wall. In the present study, we verified this hypothesis by investigating the proliferative ability and count of EPCs in peripheral blood of patients with unruptured intracranial aneurysms (UIAs). Twenty-four patients with UIAs (UIA group) and 24 negative controls (control group) were included in this study. Peripheral blood monocytes (PBMCs) were harvested and selectively cultured. The colony-forming ability of cultured cells was analyzed and the biological functions were examined by testing the adsorption of ulex europaeus agglutinin-1 labeled by fluorescein isothiocyanate and acetylated low-density lipoprotein internalization. The migratory and adhesive ability of cultured EPCs were assessed. In vitro cultured PBMCs were identified as EPCs by examining surface markers CD34, CD133 and vascular endothelial growth factor receptor 2 using flow cytometry. EPCs from UIA group possessed significantly decreased proliferative, migratory and adhesive capacities compared with EPCs from control group. Furthermore, EPCs count in UIA group was significantly decreased. Collectively, these results indicated that the circulating EPCs of UIA patients may be involved in intracranial aneurysm repair and remodeling.     

Potential implications of vascular wall resident endothelial progenitor cells

A rapidly increasing body of data suggests an essential role of endothelial progenitor cells (EPCs) in vascular regeneration, formation of new vessels in cardiovascular diseases and also in tumor vasculogenesis. Moreover, recent data obtained from clinical studies with anti-angiogenic drugs in tumor therapy or with pro-angiogenic stimuli in ischemic disorders implicate a predictive role of the number of EPCs circulating in the peripheral blood in monitoring of these diseases. However, there is still some controversial data regarding the relevance of the EPCs in vascular formation depending on models used and diseases studied. One of the essential prerequisites for a better understanding of the whole contribution of EPCs to vascular formation in adult, a process called postnatal vasculogenesis, is to identify their exact sources. We could recently discover the existence of EPCs in a distinct zone of the vascular wall of large and middle sized adult blood vessels and showed that these cells are capable to differentiate into mature endothelial cells, to form capillary sprouts in arterial ring assay and to build vasa vasorum-like structures within the vascular wall. They also can be mobilized very rapidly from the vascular wall by tumor cells. This review will discuss the functional implications of these vascular wall resident endothelial progenitor cells (VW-EPCs) in relation to those of EPCs circulating in peripheral blood or derived from the bone marrow in cardiovascular and neoplastic diseases.     

Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation

The endothelial progenitor cells (EPCs) are responsible for postnatal vasculogenesis in physiological and pathological neovascularization and have been used for attenuating ischemic diseases. However, EPCs from umbilical cord blood (CB) were not well understood and the homing mechanisms of EPCs remain unclear. To determine the potential application of CB-derived EPCs, we established a culture system to induce the differentiation of CB cells into EPCs. Purified CB CD133(+) cells proliferated and, after further vascular endothelial growth factor receptor 2 (VEGFR-2) antibody purification, differentiated into EPCs expressing endothelial markers, such as VE-cadherin, VEGFR-2, CD31, von Willebrand factor (vWF) and Weibel-Palade bodies. These cells could also take up acetylated lower density lipoprotein (Ac-LDL) and bind Ulex europaeus agglutinin-1 (UEA-1).When expanded EPCs were transplanted via tail vein into nude mice, they incorporated into capillary networks in ischemic hindlimb, augmented neovascularization, and improved ischemic limb salvage. In addition, in ischemic tissue, there were elevated expressions of VEGF and stromal derived factor 1 alpha (SDF-1 alpha), both of which had chemotactic effect on EPCs. Moreover, P-/E-selectins was found on mouse ischemic endothelium and P-selectin glycoprotein ligand-1 (PSGL-1) on CB-derived EPCs. Neutralizing antibody against PSGL-1 blocked the homing of EPCs to ischemic area by 61%. These results demonstrate that CB CD133(+) cell-derived EPCs can be applied for therapeutic neovascularization in ischemic diseases, and reveal important roles of chemoattractants and adhesive molecules in the homing of EPCs.     

猪外周血内皮祖细胞的体外分离培养及鉴定

背景：内皮祖细胞是一种能直接分化为血管内皮细胞的前体细胞,研究表明猪的心血管解剖结构较其他低等哺乳动物更接近于人类,用其建立心血管疾病模型更具临床意义。 目的：拟在体外建立猪外周血内皮祖细胞的分离培养方法。 设计、时间及地点：细胞学体外观察,于2007-09/2008-05在美国纽约州布法罗大学心血管疾病研究中心和首都医科大学病理生理学教研室完成。 材料：12周龄健康成年猪6只,由布法罗大学实验动物中心提供。 方法：采集猪外周血,以梯度密度离心法分离血单核细胞,按3×107/孔密度接种于预先包被Ⅰ型鼠尾胶原的6孔培养板,每孔加入EGM-2内皮细胞生长培养液2 mL,贴壁法纯化扩增,取传至第2代细胞用于指标检测。 主要观察指标：采用免疫荧光染色和流式细胞仪检测内皮祖细胞表面抗原的表达,通过内吞DiI-acLDL和Matrigel血管形成实验进行细胞功能学鉴定,将第2~10代细胞按500个/孔接种后检验其克隆形成能力。 结果：猪外周血单核细胞体外培养7~10 d后,出现呈铺路石样的细胞克隆,表达内皮祖细胞表面抗原CD9,CD31,CD105,VE-Cadherin,VEGFR2和内皮源性一氧化氮合酶,部分细胞表达干细胞标志c-Kit,但不表达CD133和造血祖细胞标志CD45。细胞能内吞DiI-acLDL,在Matrigel上可以形成毛细血管样结构,且具有很强的克隆形成能力。第2,4,6,8,10代细胞克隆数及内皮祖细胞总数均基本相似（F =0.167,F=0.195,P > 0.05）。 结论：梯度密度离心联合贴壁法可成功从猪外周血中分离培养出内皮祖细胞,体外扩增后细胞增殖能力无变化。     

Relationships between vascular dysfunction, circulating endothelial progenitor cells, and psychological status in healthy subjects

Background: Although the mechanisms remain unclear, depression and mental stress are associated with endothelial dysfunction and increases risk of cardiovascular disease (CVD). Recent studies suggest that circulating endothelial progenitor cells (EPC) play an important role in endothelial repair and correlate with endothelial function. Methods: We studied the relationship between the level of circulating CD34/KDR⁺ EPCs and CD133/KDR⁺ EPCs, brachial artery flow‐mediated dilation (FMD), Depression Anxiety Stress Scales in 129 normal individuals (54 ± 10 years, 54 men) without prior CVD or diabetes. Results: Their median depression score (DS) and stress score (SS) was 4 (range 0–34) and 6 (range 0–32), respectively. As defined by the ≥75th percentile, 41 subjects (32%) had high DS (≥8) and 31 (24%) had high SS (≥14). Subjects with high DS had significantly lower FMD (5.4 ± 2.7 versus 8.0 ± 4.0%, P<0.001) and percentage of CD34/KDR⁺ EPC (1.2 ± 1.3 versus 2.0 ± 2.4%, P = 0.037), but not CD133/KDR⁺ EPC (0.56 ± 0.42 versus 0.68 ± 0.76%, P = 0.44), than those with normal DS. In contrast, there were no significant difference in FMD (6.8 ± 3.5 versus 7.3 ± 3.9%, P = 0.46), percentages of circulating CD34/KDR⁺ EPC (1.20 ± 1.28 versus 1.95 ± 2.34%, P = 0.052) and CD133/KDR⁺ EPC (0.55 ± 0.41 versus 0.67 ± 0.73%, P = 0.52) between subjects with high and normal SS. Multivariate regression analysis revealed that high DS (OR 1.08, 95% CI: 1.02–1.15, P = 0.010) and old age (OR 1.05, 95% CI: 1.01–1.10, P = 0.019), but not SS or percentage of circulating EPC, were independent predictors for decreased FMD. Conclusions: Our results demonstrated that, in subjects without significant CVD, a high DS was associated with impaired brachial FMD and depletion of circulating EPC. However, only DS, but not SS or EPC count, was an independent predictor for impaired brachial FMD. Depression and Anxiety, 2011. © 2011 Wiley‐Liss, Inc.     

绵羊骨髓来源内皮祖细胞的培养与鉴定*☆

背景：当今,组织工程化静脉瓣的研究刚刚起步,种子细胞的选择是其关键,内皮祖细胞可以作为组织工程化静脉瓣体外构建理想的种子细胞。 目的：通过体外培养与鉴定绵羊骨髓来源内皮祖细胞,探讨绵羊骨髓来源内皮祖细胞培养方法,为绵羊组织工程静脉瓣种子细胞选择提供实验基础。 方法：绵羊骨髓经条件培养基进行选择培养获取骨髓单个核细胞,传代扩增后用磁珠分选出CD133+细胞再培养,流式细胞检测确认分前细胞CD133的表达情况;分选传代后绘制细胞生长曲线观察细胞生长能力,用免疫细胞化学检测细胞特异性分子CD133,D34,VWF的表达情况;FITC标记BS-1-lectin和DiI标记ac-LDL标记细胞检测细胞吞噬功能。 结果与结论：绵羊骨髓单个核细胞原代培养2 d细胞开始贴壁,7 d细胞完全融合,传代后第2天进入对数生长期,传代3~5 d形成为典型的铺路石样,传代后第7 d细胞进入增殖平台期;细胞传代后磁珠分选率为12.6%,流式细胞仪检测显示CD133阳性率为12.64%;免疫细胞化学检测显示细胞呈CD133、CD34、血管性血友病因子阳性表达。细胞能同时吞噬FITC-labeled BS-1-lectin,DiI-ac-LDL阳性率达85.3%。证实实验成功地从绵羊骨髓单个核细胞中分离培养出内皮祖细胞。