缺血性脑血管病患者颈动脉粥样硬化程度与循环内皮祖细胞的相关性分析

目的探讨缺血性脑血管病患者颈动脉粥样硬化程度与循环内皮祖细胞(EPCs)的相关性。方法根据CD133和KDR标记,采用流式细胞仪检测缺血性脑血管病患者(包括TIA、急性脑梗死、颈动脉粥样硬化)和健康体检者外周血内皮祖细胞(EPCs)数量变化。结果缺血性脑血管病患者EPCs数量较对照组明显减少(P<0.01);随着颈动脉粥样硬化程度加重,EPCs数量呈降低趋势,颈动脉重度狭窄与轻度和中度狭窄相比有差异(P<0.05);急性脑梗死和TIA患者较单纯颈动脉粥样硬化患者EPCs数量明显增加(P<0.01)。结论缺血性脑血管病患者EPCs与颈动脉粥样硬化程度呈负相关,可反映颈动脉粥样硬化的程度;急性缺血可能会增加EPCs的动员。     

通心络对颈动脉粥样硬化斑块患者内皮祖细胞增殖能力的影响

目的探讨通心络胶囊对颈动脉粥样硬化斑块患者内皮祖细胞（endothelial progenitor cells,EPCs）功能的影响。方法选择颈动脉粥样硬化斑块（软斑和/或混合斑）患者20例,分别于通心络治疗前和治疗后1个月诱导分化其外周血来源的EPCs,培养第7天测定EPCs的增殖能力、细胞和集落计数并进行对比。结果通心络治疗前EPCs增殖能力为（0.193&#177;0.037）,细胞计数为（60.24&#177;11.36）,集落计数为（2.50&#177;0.41）;治疗后EPCs增殖能力为（0.260&#177;0.044）,细胞计数为（80.16&#177;14.01）,集落计数为（4.22&#177;0.79）,治疗前后相比差异具有统计学意义（P〈0.05）。结论通心络可以增强颈动脉粥样硬化斑块患者EPCs的增殖能力,增加EPCs集落和细胞数目。     

sEHi对颈动脉狭窄患者内皮祖细胞增殖及PI3K/Akt信号通路的影响

目的 探讨可溶性环氧化物水解酶抑制剂(sEHi)AUDA 调控颈动脉狭窄(CS)患者外周血内皮祖细胞(EPCs)增殖的分子机制。方法 从CS患者外周血分离、培养内皮祖细胞,收集培养至第7 d的细胞,分为未处理组、AUDA组、PI3K抑制剂(LY294002)组和AUDA+ LY294002组,取无颈动脉狭窄患者的EPCs作为对照组,MTT法检测EPCs的增殖能力,Western blot法检测EPCs Akt磷酸化的水平。结果 对照组EPCs增殖能力较未处理组增强,AUDA组较未处理组、AUDA+ LY294002组、LY294002组EPCs增殖能力增强,未处理组、AUDA+ LY294002组较LY294002组内皮祖细胞增殖能力增强; Western blot结果显示AUDA可以促进EPCs P-Akt蛋白的表达,而LY294002可以抑制上述作用。结论 AUDA可能通过活化PI3K /Akt信号通路来促进内皮祖细胞的增殖。     

sEHi对颈动脉狭窄患者晚期内皮祖细胞功能的影响

目的研究不同浓度可溶性环氧化物水解酶抑制剂(soluble epoxide hydrolase inhibitor,s EHi)AUDA对颈动脉狭窄(carotid stenosis,CS)患者外周血来源的晚期内皮祖细胞(late endothelial progenitor cell,late EPC)的影响。方法入选研究对象60例,分成颈动脉狭窄组(n=35)和对照组(n=25)。密度梯度离心法,从外周血获取单个核细胞培养至21 d后鉴定内皮祖细胞;以不同浓度AUDA(0,0.1,1,10μmol/L)和晚期EPC共培养24 h,分别采用MTT法,黏附能力测定实验和Transwell小室来观察其增殖,黏附,迁移能力。同时采用Western blot法观察AUDA处理后其VEGF的表达。结果体外培养21 d时,细胞呈典型长梭形,21 d时,呈铺路石样,并可摄取FITCUEA-I和Dil-ac LDL。与对照组相比,CS患者late EPC的增殖,黏附和迁移能力均显著下降(P0.05);与处理前(0umol/L)相比,AUDA呈剂量依赖性地增强CS患者late EPC增殖,黏附和迁移能力并促进CS患者late EPC表达VEGF。结论 s EHi具有促进late EPC增殖,黏附和迁移等功能的作用,其有望成为一类治疗CS的新型药物。     

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.     

人脐带血内皮祖细胞与血管内皮生长因子和碱性成纤维细胞生长因子共培养及其增殖和迁移能力

背景：目前组织工程心脏瓣膜再内皮化种子细胞主要来源于成熟内皮细胞,内皮祖细胞作为内皮细胞的前体细胞越来越受到人们的关注。 目的：分离和扩增人脐带血内皮祖细胞,观测其体外生物学特性。 方法：密度梯度离心法分离新鲜的脐血中单个核细胞,在含血管内皮生长因子和碱性成纤维细胞生长因子的培养液中培养扩增,通过形态学、免疫荧光和流式细胞仪等对贴壁细胞进行鉴定;并与脐静脉内皮细胞进行增殖和迁移能力比较。 结果与结论：随着培养和诱导时间延长,贴壁细胞形态发生明显的改变,从小圆形变成梭形,逐渐分化成典型成熟内皮细胞的鹅卵石样形态,并可形成特征性的克隆;体外诱导7 d后90%以上贴壁细胞呈Dil-ac-LDL和FITC-UEA-I双阳性;贴壁细胞流式细胞仪分析显示：培养7 d的细胞VEGFR-2、CD34和CD133表达分别占(77.4±4.9)%、(52.4±6.6)%和(19.4±2.1)%,培养28 d的细胞VEGFR-2和CD34表达分别占(81.1±7.4)%和(7.6±3.1)%,而未检测到CD133表达;人内皮祖细胞增殖和迁移能力明显高于人脐静脉内皮细胞(P < 0.05),并且细胞数量可扩增达109 L-1。结果显示用密度梯度离心法和贴壁筛选法,可从人脐带血分离、纯化内皮祖细胞;内皮祖细胞可诱导分化为内皮细胞,增殖和迁移能力都很强。     

缺血性脑血管病与颈动脉粥样硬化及其危险因素的关系

目的探讨缺血性脑血管病(ICVD)与颈动脉粥样硬化及其危险因素的关系。方法对186例ICVD患者与194例非脑血管病患者和正常体检者(对照组)行颈部血管超声检查和血液生化检查;比较两组间的颈动脉硬化程度及脑卒中危险因素的差异。结果ICVD组年龄[(69±7)岁]和患有高血压(66.1%)、糖尿病(53.4%)、代谢综合征患者(44.6%)的比率非常明显高于对照组[(61±5)岁、48.8%、15.2%、12.9%](均P<0.001)。ICVD组颈动脉粥样硬化分级计分≥2分(斑块发生率)、≥3分(血管狭窄发生率)分别为69.3%、20.4%,明显高于对照组的33.5%和5.1%(均P<0.05)。结论颈动脉粥样硬化是ICVD的危险因素之一;各种危险因素的聚集对ICVD的发生起重要作用。     

参麦注射液对人外周血内皮祖细胞部分生物学的影响

目的观察参麦注射液对体外培养外周血来源内皮祖细胞（endothelial progenitor cells，EPCs）数量及增殖、迁移、粘附功能的影响。方法密度梯度离心法获取人外周血单个核细胞，FITC-荆豆凝集素I（FITC-UEA-1）、DiL-乙酰化低密度脂蛋白（DiL-acLDL）荧光双染鉴定；单个核细胞培养4d后进行实验分组，分为对照组和参麦注射液治疗组，参麦注射液治疗组加入不同浓度参麦注射液（分别为0．01、0．1、0．15、0．3mg／L）培养48h，然后分别采用四氮唑溴盐比色法（MTT）、改良的Boyden小室和粘附能力测定来观察EPCs的增殖、迁移和粘附能力。结果不同浓度的参麦注射液对EPCs有不同作用，0．1、0．15mg／L主要表现为使EPCs功能增强，0．3mg／L时贴壁细胞数量减少及增殖、迁移、粘附功能减弱，0．01mg／L与未加药组比较无统计学意义。结论参麦注射液可影响培养EPCs的数量及其部分生物学功能。     

瑞舒伐他汀钙对颈动脉粥样硬化患者CD4+CD25+调节性T细胞的干预作用

目的 探讨CD4~+CD25~+调节性T细胞在颈动脉粥样硬化发病中的作用,研究瑞舒伐他汀钙在颈动脉粥样硬化治疗中的免疫调节作用。方法 选取颈动脉粥样硬化患者42例为治疗组,以42例健康体检者为正常对照组;治疗组随机分为瑞舒伐他汀治疗组21例和安慰剂对照组21例。利用流式细胞术检测各组CD4~+CD25~+调节性T细胞变水平。结果 (1)与正常对照组(6.67±0.79)%比较,颈动脉粥样硬化患者组CD4~+CD25~+调节性T细胞水平(3.41±0.67)%明显下降,差异具有统计学意义(P0.05)。(2)瑞舒伐他汀钙干预治疗1个月后,与治疗前(3.38±0.64)%及安慰剂组(3.42±0.56)%比较,治疗组外周血CD4~+CD25~+调节性T细胞表达水平(6.02±0.84)%显著性升高,差异具有统计学意义(P0.05)。结论 颈动脉粥样硬化患者外周血CD4~+CD25~+调节性T细胞水平降低,瑞舒伐他汀钙可能通过上调CD4~+CD25~+调节性T细胞水平起到免疫调节作用,从而达到延缓颈动脉粥样硬化的进展。     

脑梗死患者外周血调节性B细胞水平及其与颈动脉粥样硬化严重程度的相关性

目的探讨脑梗死患者外周血调节性B细胞(Bregs)水平变化及其与颈动脉粥样硬化严重程度的相关性。方法选取2017-09-01—2018-05-01十堰市太和医院收治的动脉粥样硬化性脑梗死患者50例,并选择同期健康体检者50名为对照组。采用流式细胞术(FCM)检测外周血B细胞中CD19+CD5+CD1d+B细胞(Bregs)所占B细胞的比例;采用ELISA法测定Bregs培养液上清中白细胞介素-10(IL-10)及转化生长因子-β1(TGF-β1)水平。分析脑梗死患者外周血Bregs水平变化以及其与颈动脉粥样硬化程度的关系。结果脑梗死患者外周血Bregs水平及其分泌的IL-10、TGF-β1水平较对照组降低(P0.05);随颈动脉粥样硬化严重程度加重,脑梗死患者外周血Bregs水平及IL-10、TGF-β1水平逐渐降低(均P0.05)。结论动脉粥样硬化性脑梗死患者外周血Bregs水平低于健康人;Bregs水平与动脉粥样硬化性脑梗死患者颈动脉粥样硬化严重程度密切相关;外周血Bregs水平的降低以及功能的丧失参与了脑梗死的发生和发展,其机制可能与机体免疫平衡被打破有关。     

补阳还五汤对外周血内皮祖细胞数量和功能的影响

背景：现代医学研究表明,内皮祖细胞和补阳还五汤在治疗缺血性疾病上具有相似的功能,两者之间是否存在着一定的联系？ 目的：观察补阳还五汤对外周血内皮祖细胞数量和功能的影响。 方法：采用密度梯度离心法分离培养兔外周血内皮祖细胞,经Dil-ac-LDL和FITC-UEA-I双染色和表面抗原的免疫组织化学检测鉴定后,将贴壁细胞随机分为4组,分别用基础培养基以及含有20,50,100 g/L补阳还五汤的EBM-2培养基进行细胞培养72 h。检测细胞形态及计数,再采用四甲基偶氮唑盐微量酶反应比色法、Transwell小室、黏附功能检测、一氧化氮检测及血管内皮生长因子免疫细胞化学分析来评定其增殖、迁移、黏附、分泌一氧化氮和表达血管内皮生长因子的能力。 结果与结论：不同质量浓度补阳还五汤均能增加内皮祖细胞的数量,提高内皮祖细胞增殖、黏附、迁移和分泌一氧化氮的能力(P < 0.05),药物质量浓度在50 g/L时影响最为显著(P < 0.01),而对血管内皮生长因子表达的影响较微弱。提示补阳还五汤能增加内皮祖细胞的数量,提高内皮祖细胞的增殖、迁移和黏附能力,并可能诱导晚期内皮祖细胞的分化。     

Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.

Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G>C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells (P<0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner (P<0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects (P<0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.     

Changes and function of circulating endothelial progenitor cells in patients with cerebral aneurysm

Endothelial dysfunction is a trigger for the formation of cerebral aneurysm (CA). The circulating endothelial progenitor cell (EPC) plays an important role in postnatal vasculogenesis and reduction of endothelial injury. In this study, we tested the hypothesis that decreased number and impaired function of circulating EPCs correlate with CA formation in patients. Blood circulating EPCs were identified by flow cytometry. The level of plasma vascular endothelial growth factor (VEGF) was measured by ELISA. Circulating EPCs from patients (n = 27) were cultured in vitro, and the function of EPCs was evaluated by cell migration and senescence-associated β-galactosidase activity. The number of circulating EPCs was significantly decreased in both unruptured and ruptured CA patients compared with healthy control subjects. Impaired migratory capacity and elevated cellular senescence of cultured EPCs were observed in patients with CA (ruptured and unruptured). The percentages of EPC senescence in patients with CAs were significantly and negatively correlated with the number of circulating EPCs. In addition, there were higher levels of plasma VEGF in CA patients compared with healthy control subjects. Our results show that the numbers and functions of circulating EPCs are reduced in patients with CAs. These findings suggest that the decreased number and impaired function of circulating EPCs in CA patients may contribute to the pathophysiological process of aneurysm formation.     

Cilostazol enhances integrin-dependent homing of progenitor cells by activation of cAMP-dependent protein kinase in synergy with Epac1

Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 μM) stimulated up-regulation of integrins β1, α1, and αv as well as 8-pCPT-2'-O-Me-cAMP (100 μM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 μM) and GGTI-298 (Rap1 inhibitor, 20 μM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N(6)-benzoyl-cAMP (100 μM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin β1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease.     

Circulating Endothelial Progenitor Cells in Cerebrovascular Disease

Stroke is associated with high disability and mortality burdens worldwide, but there are few effective and widely available therapies. There is therefore a need to develop treatments that promote the repair and regeneration of ischemic brain tissue. In this regard, a population of adult stem cells-called endothelial progenitor cells (EPCs)-has been identified in peripheral blood that could provide novel approaches in regenerative medicine for curing patients with acute ischemic stroke. There is accumulating evidence that EPCs can repair damaged endothelia and attenuate the development and progression of atherosclerosis. Also, EPCs can be recruited in response to acute ischemic events and participate in reparative vasculogenesis. Most studies related to EPCs have involved patients with cardiovascular diseases, and there is emerging evidence that EPCs represent a risk marker and a potential therapeutic agent in cerebrovascular disease. Here we review the characteristics and biology of EPCs in cerebrovascular disease and discuss the challenges that must be addressed to clarify the role and therapeutic applicability of EPCs in cerebrovascular disease.     

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.     

Serial measurement of surface expressions of CD63, P-selectin and CD40 ligand on platelets in atherosclerotic ischemic stroke. A possible role of CD40 ligand on platelets in atherosclerotic ischemic stroke

BACKGROUND: Platelet activation is a key step in the progression of atherosclerosis. The CD40 ligand (CD40L) on platelets may be a critical factor to develop the acute vascular events from atheroma. METHODS: To determine the role of CD40L on platelets in atherosclerotic ischemic stroke, we serially measured the expressions of CD63, P-selectin and CD40L on platelets in patients with atherosclerotic ischemic stroke (n = 25) and compared them with those in patients with asymptomatic carotid stenosis (n = 20) and in normal subjects (n = 24). RESULTS: The expressions of CD63 and P-selectin on platelets were significantly higher in patients with atherosclerotic ischemic stroke (n = 25) than in normal subjects (n = 24). The extents of surface expressions of CD63 and P-selectin on platelets showed no significant differences between atherosclerotic ischemic stroke and asymptomatic carotid stenosis. However, the CD40L expression on platelets was significantly higher in atherosclerotic ischemic stroke when compared to that in asymptomatic carotid stenosis. CONCLUSIONS: In our data, among the population with large artery atherosclerosis, the patients with symptomatic ischemic events showed a significantly elevated expression of CD40L on platelets compared to those without ischemic events. Therefore, the upregulation of CD40L on platelets may be a specific marker of platelet activation to provoke ischemic stroke from large artery atherosclerosis.     

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.