兔骨髓源血管内皮祖细胞的体外培养与鉴定

目的 体外分离、培养、扩增血管内皮祖细胞(EPCs),观察EPCs生长分化过程并对其生物学特性进行鉴定.方法 采用密度梯度离心法从兔骨髓中提取单个核细胞,贴壁筛选法分离EPCs,于添加了Singlequotes的EBM-2培养液中扩增培养,对培养10d的细胞进行免疫荧光及免疫组织化学分析.结果 培养4 d,光电显微镜可见细胞集落形成,梭形贴壁细胞从集落中央以放射状向外周生长;培养7～10 d,细胞集落相互连接,呈典型的"鹅卵石"样外观;2周左右可见细胞排列成条索状结构.贴壁细胞呈DIL-ac-LDL及FITC-UEA-1双荧光阳性,阳性率为(65.0±4.0)%;贴壁细胞表达vWF,VEGFR-2和VE-cadherin的阳性率分别为(90.0±2.1)%,(77.0±4.1)%和(78.1±8.2)%.结论 骨髓中富含EPCs,成功建立了一整套骨髓源EPCs的分离、体外扩增培养的方法,且操作简便具有较好的可重复性.     

黄芪提取物对大鼠骨髓源性内皮祖细胞的作用

摘要： 目的 探讨黄芪提取物对大鼠骨髓源性内皮祖细胞 （EPCs） 黏附、 迁移、 活力、 血管形成能力及内皮型一氧 化氮合成酶 （eNOS） 表达的影响。方法 体外培养、 分离和鉴定 EPCs, 设 10-4、 10-3、 10-2 g/L 黄芪提取物组和对照组。 倒置显微镜下观察并比较各组 EPCs 黏附、 迁移、 血管形成能力的差异; MTT 法检测 EPCs 的活力变化; RT-PCR 法检 测 EPCs 中 eNOS mRNA 的表达;Western blot 检测 EPCs 中 eNOS 蛋白的表达。结果 与对照组相比, 不同浓度黄芪 提取物组促 EPCs 黏附、 迁移、 血管形成能力均显著增强, 且呈浓度依赖性(F 值分别为 15.256、 13.633、 97.549, 均 P < 0.05); EPCs 的活力显著增加, 呈时间 （F 时间=9.755） 和浓度依赖性 （F 组间=10.018）; 且 EPCs 中 eNOS mRNA 和蛋白的表 达水平均明显升高, 呈浓度依赖性(F 值分别为 56.356、 77.125, 均 P < 0.05)。结论 黄芪提取物具有调控 EPCs 促血 管新生的作用, 该作用可能与上调eNOS 的表达水平密切相关。     

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。     

SDF 1α对大鼠骨髓源性内皮祖细胞克隆形成能力及增殖的影响

目的观察基质细胞衍生因子-1α（SDF-1α）对体外培养的大鼠骨髓源性内皮祖细胞（EPCs）动员和增殖的影响。方法微孔法获取大鼠骨髓EPCs并采用荧光显微镜和流式细胞术鉴定EPCs特异性标记物。不同浓度SDF-1α处理EPCs后,采用细胞培养、MTT检测EPCs的克隆形成、细胞增殖。结果通过MTT法检测,对照组与（1、10、100μg/L）SDF-1α处理组的OD值分别为0.311±0.054、0.587±0.072、0.813±0.056、1.029±0.078,通过统计学方法分析,对照组与SDF-1α处理组比较差异均有统计学意义（n=5,P〈0.05）;100μg/LSDF-1α处理组次级内皮祖细胞集落单位数目是对照组近3倍（4.67±1.577比14.33±3.055,n=5,P〈0.01）。结论SDF-1α剂量依赖性地促进EPCs增殖,并显著增强EPCs克隆形成能力。     

内皮祖细胞的迁移及对血管修复作用的研究进展

血管内皮损伤及其修复功能发生障碍在心血管疾病的发生发展过程中起着重要作用,内皮祖细胞是干细胞的一种亚型,为内皮细胞的前体细胞,在成年机体内主要定居在骨髓干细胞池中,具有高增殖潜能.多种因素可刺激EPCs从骨髓动员到外周血液,在细胞因子等作用下归巢到血管损伤部位并分化为成熟内皮细胞.本文就骨髓EPCs从动员到分化为内皮细胞过程中不同标志物的表达及可能存在的分子信号机制进行综述.     

雌激素对糖尿病大鼠内皮祖细胞功能的影响及其机制研究

目的 探索雌激素对糖尿病大鼠内皮祖细胞(EPCs)功能的影响及其可能的作用机制。方法 取健康Wistar大鼠骨髓提取EPCs并采用流式细胞仪和荧光显微镜鉴定。大鼠给予链脲佐菌素诱导为糖尿病模型,提取正常大鼠和糖尿病大鼠骨髓EPCs并培养,糖尿病大鼠EPCs体外给予雌激素10 nmol/L孵育24 h。检测EPCs增殖和功能;测定EPCs中锰超氧化物歧化酶(MnSOD)水平和NO水平及上清液中凝血酶敏感蛋白-1(TSP-1)蛋白水平。结果 与对照组比较,糖尿病EPCs的细胞增殖能力、迁移能力和小管形成功能受损(P<0.01),而雌激素体外干预后细胞增殖能力、迁移能力和小管形成功能均得到改善(P<0.01);糖尿病EPCs中MnSOD水平和NO水平明显下调,上清液中TSP-1蛋白水平升高(P<0.01);雌激素孵育能逆转上述改变(P<0.01)。结论 雌激素能改善糖尿病大鼠EPCs迁移能力和小管形成功能,作用机制可能与其降低糖尿病EPCs内的氧化应激及下调TSP-1的表达相关。     

产前尼古丁暴露对子代大鼠血管内皮祖细胞的影响

目的 明确产前尼古丁暴露对子代大鼠血管内皮祖细胞的影响,并探讨该效应是否存在性别差异、是否可持续作用至日后成年期。方法 孕Sprangue-Dawley大鼠20只随机分为两组,产前尼古丁暴露（PNE）组与正常对照组各10只,于孕前7d皮下植入渗透微泵（2ML4型）,其中PNE组渗透微泵携带102mg/mL尼古丁,而正常组携带生理盐水。分别于子代大鼠1、3、6、12月龄,取外周血采用流式细胞术测定内皮祖细胞（EPCs）数量。结果 流式细胞术检测子代大鼠外周血EPCs:1月龄雌性子代,PNE组较正常组显著减少（P <0.05）、3月龄差异无统计学意义（P> 0.05）、6月龄显著增多（P <0.05）、12月龄差异无统计学意义（P> 0.05）;1、3、6、12月龄雄性子代PNE组与正常组相比差异无统计学意义（P> 0.05）。结论PNE可诱导雌性子代SD大鼠外周血循环内皮祖细胞显著减少,其作用可持续至成年期,而对雄性子代无明显作用呈性别依赖性差异特征。     

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.     

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。     

大鼠脾源性内皮祖细胞培养及鉴定

目的 培养、鉴定大鼠脾源性内皮祖细胞.方法 机械损伤大鼠脾脏,用密度梯度离心法收集单个核细胞层,富含胎牛血清EMDM培养液培养,观察细胞形态,将培养第7天内皮祖细胞与DiI标记的乙酰化低密度脂蛋白37℃孵育4h,以检测内皮祖细胞对Dil-乙酰化低密度脂蛋白的摄取,再用20 g/I,多聚甲醛固定细胞10 min,固定后用...     

Decreased mobilization of endothelial progenitor cells contributes to impaired neovascularization in diabetes

• 1 Circulating bone marrow (BM)‐derived endothelial progenitor cells (EPCs) play an important role in neovascularization. In the present study, we investigated the mechanisms underlying the reduction in circulating EPCs in a mouse model of diabetes induced by streptozotocin.
• 2 Compared with non‐diabetic controls, diabetic mice had reduced circulating EPCs (0.59 ± 0.11 vs 0.94 ± 0.21%, respectively; P < 0.01) and increased plasma endothelial microparticles (18 642 ± 6809 vs 5692 ± 1862/mL, respectively; P < 0.01). In a mouse bone marrow (BM) transplantation model, increased adhesion of transplanted BM cells to aortas of diabetic mice was observed compared with control (900 ± 194 vs 431 ± 109 cells/mm², respectively; P < 0.01).
• 3 Following hindlimb ischaemia, diabetic mice exhibited suppressed EPC mobilization, a reduction in the expected increase in capillary density and suppressed restoration of transcutaneous oxygen pressure in the ischaemic tissue. Diabetic mice also showed impaired ischaemia‐induced upregulation of vascular endothelial growth factor (VEGF), hypoxia‐inducible factor (HIF)‐1α and interleukin‐1β, an exaggerated increase in matrix metalloproteinase (MMP)‐2 and ‐9 and a suppressed increase in tissue inhibitor of matrix metalloproteinase (TIMP)‐1. On multivariate analysis, VEGF expression was the only independent factor related to circulating EPC count.
• 4 In conclusion, the data indicate that the decrease in basal circulating EPCs in diabetes may be attributable, in part, to consumptive loss of EPCs due to increased endothelial damage. Impairment of ischaemia‐induced EPC mobilization in the diabetic mouse model is associated with altered HIF‐1α/VEGF and MMP/TIMP regulation and represents a novel mechanism underlying defective postischaemic neovascularization in diabetes.

     

Progenitors in motion: mechanisms of mobilization of endothelial progenitor cells

Endothelial progenitor cells are a population of bone marrow-derived mononuclear cells thought to engage in endothelial repair and hence are considered potential therapeutic agents in many pathological conditions. The mechanism of their exit from the bone marrow to the circulation and damaged tissues, termed mobilization, has not been fully elucidated. Despite this, several pharmacological interventions have been shown to influence mobilization of these specialized cells. Here we review the current understanding of their mobilization.     

Effects of electroacupuncture on endothelial function and circulating endothelial progenitor cells in patients with cerebral infarction

This study evaluated the effects of electroacupuncture (EA) on endothelial function and endothelial progenitor cells (EPC) in patients with cerebral infarction. In a randomized, placebo‐controlled, crossover study, 20 patients with cerebral infarction were randomized into two treatment groups: EA or placebo. Before and after each intervention, pulse amplitude tonometry (PAT) was used to assess endothelial function and peripheral blood was analyzed for the number of EPCs. Circulating EPCs were quantified by flow cytometry as CD45^lowCD34⁺KDR2⁺ cells. Plasma vascular endothelial growth factor (VEGF) and interleukin (IL)‐10 levels were measured. Seven days later, crossover was performed on each group, with each group receiving the other treatment using the same protocol. The PAT hyperemia ratio ranged from 1.57 ± 0.41 to 2.04 ± 0.51 after EA, representing a significant improvement (P = 0.002); however, there was no improvement in the placebo group (P = 0.48). Circulating EPCs, as measured by flow cytometry, increased to 110.6 ± 74.3/100 μL in the EA group (P = 0.001) but did not change in the placebo group (45.9 ± 35.3/100 μL, P = 0.08). The increases in the number of EPCs and the PAT ratio after treatment were correlated (r = 0.78, P < 0.001). Plasma VEGF levels increased with EA compared to baseline (261.2 ± 34.0 vs 334.9 ± 80.5 pg/mL, P = 0.003). The number of circulating EPCs was positively correlated with plasma levels of VEGF (r = 0.50, P = 0.02). In conclusion, EA induced improvement of EPC levels and the PAT ratio in patients with cerebral infarction.     

改善糖尿病内皮祖细胞功能的研究进展

内皮祖细胞（EPC）为来源于骨髓及外周血中的干细胞,能定向增殖分化为内皮细胞,参与血管损伤后的修复。研究显示,改善糖尿病EPC功能,可以预防和降低糖尿病并发症的发生。追踪近年来改善EPC功能的研究进展,就其对糖尿病并发症的预防和改善作用做一综述,以期为糖尿病的治疗提供新思路。     

内皮祖细胞的鉴定及培养方法

近10年来,内皮祖细胞(endothelial progenitor cells,EPCs)的研究受到越来越多的关注。研究发现,循环血液中EPCs储量的改变伴随着心血管疾病的不同表现。因此,EPCs作为心血管疾病的生物标志物及修复受损血管的一种细胞疗法被广泛研究。该文对1997年至今大多数研究中EPCs的鉴定及培养方法做了简要综述。     

Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells

BACKGROUND AND PURPOSE

Current methods used to treat critical limb ischaemia (CLI) are hampered by a lack of effective strategies, therefore, therapeutic vasculogenesis may open up a new field for the treatment of CLI. In this study we investigated the ability of the DPP-4 inhibitor, sitagliptin, originally used as a hypoglycaemic agent, to induce vasculogenesis in vivo.

EXPERIMENTAL APPROACH

Sitagliptin were administered daily to C57CL/B6 mice and eGFP transgenic mouse bone marrow-transplanted ICR mice that had undergone hindlimb ischaemic surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neovasculogenesis and circulating levels of endothelial progenitor cells (EPCs) respectively. Cell surface markers of EPCs and endothelial NOS (eNOS) in vessels were studied.

KEY RESULTS

Sitagliptin elevated plasma glucagon-like peptide-1 (GLP-1) levels in mice subjected to ischaemia, decreased plasma dipeptidyl peptidase-4 (DPP-4) concentration, and augmented ischaemia-induced increases in stromal cell-derived factor-1 (SDF-1) in a dose-dependent manner. Blood flow in the ischaemic limb was significantly improved in mice treated with sitagliptin. Circulating levels of EPCs were also increased after sitagliptin treatment. Sitagliptin also enhanced the expression of CD 34 and eNOS in ischaemic muscle. In addition, sitagliptin promoted EPC mobilization and homing to ischaemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice.

CONCLUSION AND IMPLICATIONS

Circulating EPC levels and neovasculogenesis were augmented by the DPP-4 inhibitor, sitagliptin and this effect was dependent on an eNOS-related pathway in a mouse model of hindlimb ischaemia. The results indicate that oral administration of sitagliptin has therapeutic potential as an inducer of vasculogenesis.     

普伐他汀对人内皮祖细胞一氧化氮合成的影响

目的观察普伐他汀对人内皮祖细胞（EPCs）一氧化氮（NO）合成的影响。方法密度梯度离心法获取外周血单个核细胞,培养7d后,收集贴壁细胞并分别加入普伐他汀,10μmol/L及100μmol/L干预48h,免疫组化、荧光显微镜和流式细胞仪鉴定EPC,用RT-PCR方法测定对细胞内皮型一氧化氮合酶（eNOS）mRNA表达的影响,并用硝酸还原酶法测定培养液中一氧化氮（NO）的水平。结果普伐他汀组的人内皮祖细胞eNOS mRNA的表达、NO的合成明显增加。结论普伐他汀可增加人内皮祖细胞eNOS mRNA的表达和NO的合成     

The effect of acute exposure to coarse particulate matter air pollution in a rural location on circulating endothelial progenitor cells: results from a randomized controlled study

Abstract

Context: Fine particulate matter (PM) air pollution has been associated with alterations in circulating endothelial progenitor cell (EPC) levels, which may be one mechanism whereby exposures promote cardiovascular diseases. However, the impact of coarse PM on EPCs is unknown.

Objective: We aimed to determine the effect of acute exposure to coarse concentrated ambient particles (CAP) on circulating EPC levels.

Methods: Thirty-two adults (25.9?±?6.6 years) were exposed to coarse CAP (76.2?±?51.5?μg?m^?3) in a rural location and filtered air (FA) for 2 h in a randomized double-blind crossover study. Peripheral venous blood was collected 2 and 20 h post-exposures for circulating EPC (n?=?21), white blood cell (n?=?24) and vascular endothelial growth factor (VEGF) (n?=?16–19) levels. The changes between exposures were compared by matched Wilcoxon signed-rank tests.

Results: Circulating EPC levels were elevated 2 [108.29 (6.24–249.71) EPC?mL^?1; median (25th–75th percentiles), p?=?0.052] and 20 h [106.86 (52.91–278.35) EPC?mL^?1, p?=?0.008] post-CAP exposure compared to the same time points following FA [38.47 (0.00–84.83) and 50.16 (0.00–104.79) EPC?mL^?1]. VEGF and white blood cell (WBC) levels did not differ between exposures.

Conclusions: Brief inhalation of coarse PM from a rural location elicited an increase in EPCs that persisted for at least 20 h. The underlying mechanism responsible may reflect a systemic reaction to an acute “endothelial injury” and/or a circulating EPC response to sympathetic nervous system activation.