流体剪切应力对晚期内皮祖细胞生物学功能的影响

目的 研究流体剪切应力处理对晚期内皮祖细胞（endothelial progenitor cells,EPCs）体外及体内生物学功能的影响。 方法 密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2MV进行体外培养。以3~4代的EPCs,即晚期EPCs为靶细胞,对其施以1.2 Pa剪切应力处理。采用EdU标记技术、黏附能力测定实验、改良的Boyden小室、Annexin V/PI、β 半乳糖苷酶检测法、Matrigel法、荧光定量RT PCR等方法分别检测剪切应力对晚期EPCs增殖、黏附、迁移、凋亡、衰老、体外成血管及VEGF mRNA表达等生物学功能的影响。应用大鼠颈动脉损伤模型及细胞原位移植等实验手段检测剪切应力预处理对晚期EPCs修复受损内皮的影响。结果1.2 Pa剪切应力处理可不同程度提高晚期EPCs的增殖、黏附、迁移及成血管能力（P＜0.01）,上调VEGF的基因表达,抑制晚期EPCs的衰老及凋亡（P＜0.01）;移植经剪切应力预处理的晚期EPCs可加速损伤内皮的修复,减缓内膜的增生。结论 流体剪切应力可改善晚期EPCs的功能活性,提高晚期EPCs修复损伤血管内皮的能力, 这为EPCs的临床应用及剪切应力介导的细胞疗法提供了实验依据。     

大鼠骨髓源性血管内皮祖细胞体外培养分化及鉴定

目的：探索大鼠骨髓源性血管内皮祖细胞（EPCs）的培养、诱导分化及鉴定方法。方法：冲洗大鼠骨髓腔,梯度密度离心法获得单个核细胞,内皮细胞培养液EGM-2MV培养,通过细胞形态,免疫组化和免疫荧光检测CD34、VEGFR-2、vWF,CD133,摄取Dil-ac-LDL和结合FITC-Lectin-UEA-1,超微结构及染色体核型分析进行鉴定。结果：新分离的骨髓单个核细胞呈圆形,大小不一;48h后部分细胞开始贴壁,呈梭形、纺锤形或不规则形;4～8d呈细胞集落或线状排列;9～11d接近融合,呈典型鹅卵石样外观。贴壁细胞CD34、CD31、VEGFR-2、vWF、CD133均表达阳性并呈动态变化,能够摄取Dil-ac-LDL和结合FITC-Lectin-UEA-1,透射电镜见特征性Weible-Palade小体,能稳定保持二倍体核型。结论：本实验初步建立了一套大鼠骨髓血管内皮祖细胞分离、培养、诱导分化及鉴定方法体系。     

细胞骨架F-actin在层流剪切应力诱导EPCs内皮分化中的作用

目的探讨细胞骨架F-actin在层流剪切应力促内皮祖细胞(endothelial progenitor cells,EPCs)向内皮分化中的作用。方法对大鼠骨髓来源的EPCs施以层流剪切应力(1.2 Pa),以荧光定量RT-PCR及流式细胞术检测特异性内皮细胞标记分子vWF、CD31 mRNA及蛋白的表达来反映EPCs分化程度;以免疫荧光染色观测F-actin的排列情况;应用Ras GTPase Pull-Down方法检测Ras活性。结果层流剪切力处理后,EPCs分化标记vWF及CD31的基因及蛋白表达较静止组明显升高(P<0.05),细胞骨架F-actin发生重排,Ras活性明显增高。细胞骨架稳定剂Jasplakinolide(JAS)及细胞骨架松弛剂Cytochalasin D(CytoD)预处理均不同程度地抑制了层流剪切应力所致的细胞骨架的重排、Ras活性的上调及EPCs分化效应(P<0.05),而过表达Ras则对层流剪切应力诱导的EPCs分化有明显促进作用(P<0.05)。结论一定大小的层流剪切应力可促进EPCs向内皮细胞分化,其机制可能与层流剪切应力重塑细胞骨架F-actin,进而影响Ras活性有关;这对于揭示受损血管内皮修复的具体机制、阐明动脉粥样硬化等心血管疾病的发病机理及临床防治此类疾病具有一定的意义。     

内皮祖细胞在血管新生及血管组织工程化过程中的生物学意义

内皮祖细胞(EPCs)是能分化为成熟血管内皮细胞的祖细胞,参与了出生后的血管再生和受损内皮的修复过程。近年来围绕以EPCs作为种子细胞来促进血管新生、维持内皮功能完整并构建组织工程化血管方面展开了许多研究。本文就这方面的进展作一综述。     

外周血内皮祖细胞种植小径聚氨酯人工血管及流体切应力处理的实验研究

本研究收集健康成人骨髓单个核细胞,用血管内皮生长因子等加以诱导分化,通过荧光显微镜和荧光免疫标记等方法观察和鉴定诱导后的细胞。之后将诱导分化的内皮祖细胞种植到聚氨酯小径人工血管表面,予以15 dyn／cm2的流体切应力处理,并用扫描电镜观察。结果发现外周血单个核细胞诱导分化成为内皮祖细胞,ac- LDL及lectin抗体荧光标记阳性。扫描电镜下,未种植细胞的聚氨酯小径人工血管表面孔径大小比较适合内皮祖细胞爬行;静态种植细胞后,人工血管表面内皮祖细胞排列不整齐;切应力条件下种植细胞后,人工血管表面内皮祖细胞排列较为整齐。因此,在体外能将外周血单个核细胞诱导分化成为内皮祖细胞,内皮祖细胞是小径人工血管内皮化的理想种子细胞。流体切应力对小径聚氨酯人工血管表面内皮祖细胞的生长排列有着良好的机械塑形作用。     

脉冲电刺激对血管内皮细胞与其祖细胞黏附的影响

为探索脉冲电刺激下血管内皮细胞与内皮祖细胞(EPC)之间黏附强度的改变,诱导培养外周血EPC,荧光标记后与单层血管内皮细胞共培养,固定电压和频率为5 V和5 Hz,选择1、3、6、9 ms的脉宽对其进行干预,持续刺激24 h后检测贴壁EPC的荧光强度,以荧光比率衡量。结果显示,与对照组相比,3 ms刺激组荧光比率即显著增高,随着脉宽延长,6 ms组达到最大值,但9 ms刺激组却显著下降,提示适宜脉冲电刺激有利于血管内皮细胞与EPC之间的黏附,为电刺激促进血管新生的研究提供新的理论依据。     

人外周血内皮祖细胞的分离、培养和扩增

目的:改进从人外周血中分离、培养和体外扩增血管内皮祖细胞(EPC)的方法。方法:采用不同淋巴细胞分离液,用密度梯度离心法从外周血分离EPC;用CD34免疫磁性活化细胞分选系统(MACS)分离CD34 细胞;分别培养在包被和不包被有人纤维连接蛋白(HFN)的培养板内;采用细胞免疫化学法检测内皮细胞表面标志CD31、CD34和vWF的表达。结果:从成人外周血可分离获得EPC;不同的分离条件可影响获得EPC的数量和质量,HFN对EPC的生长有促进作用。结论:进一步改进从人外周血分离获取EPC并行体外扩增的方法,为EPC的研究奠定了基础。     

Substrate-dependent modulation of 3D spheroid morphology self-assembled in mesenchymal stem cell-endothelial progenitor cell coculture

The structural evolution of three-dimensional spheroids self-assembled from two different types of cells on selective biomaterials is demonstrated in this study. The two types of cells involved in the self-assembly are human mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). When seeded in different population ratios, they can create a variety of cellular patterns on different biomaterial substrates. When the two populations are matched in initial numbers, they are self-assembled in co-spheroids with different morphologies (i.e. randomly mixed, bumped, or concentric spheroids). The morphologies are influenced by the specific cell-substrate interaction possibly through integrin signaling, as well as a substrate-dependent regulation of heterophilic cell–cell interaction possibly through Notch signaling. In particular, the self-assembled core–shell concentric spheroids from adipose-derived MSCs and EPCs show a greater angiogenic effect in vitro. This study reveals the possibility to modulate the self-assembled morphology as well as the effect of cocultured cells by changing the cell culture substratum.     

Apelin and vascular endothelial growth factor are associated with mobilization of endothelial progenitor cells after acute myocardial infarction

This study was designed to determine the levels of early endothelial progenitor cells(EPCs),apelin,vascu-lar endothelial growth factor(VEGF) and stromal cell-derived growth factor-1(SDF-1) after acute myocardial infarction(AMI),and to investigate the relationships between these cytokines and early EPCs.Early EPCs,de-fined as CD133+,KDR+,and CD34+ cells,were quantified by flow cytometry.The levels of early EPCs and those cytokines in AMI patients were significantly different from those with coronary artery disease or controls(P < 0.05).Plasma apelin levels were inversely correlated with Gensini score and early EPCs(both P < 0.01).Early EPCs,VEGF and SDF-1 showed different patterns of changes in AMI patients during the first 24 h.The trend in the change of early EPCs was proportionally correlated with that of VEGF(P < 0.05).AMI patients exhibited in-creased early EPCs with remarkably decreased apelin levels and enhanced VEGF levels.     

兔外周血内皮祖细胞培养及HTK基因体外修饰

目的探讨兔外周血来源的内皮祖细胞(EPCs)经体外基因修饰后能否成功表达目的基因。方法分离兔外周血单个核细胞进行定向培养,观察细胞摄取DiI-Ac-LDL的能力,免疫荧光法检测EPCs的细胞表面标记。同时PCR扩增人组织激肽释放酶(HTK)基因,构建以GFP为报告基因的重组真核表达载体,转染体外培养的EPCs后,荧光显微镜下观察HTK-EGFP融合蛋白的表达,通过RT-PCR与ELISA方法检测HTK的表达水平。结果细胞培养5～6d开始出现成簇的短梭形贴壁细胞,15d后形成鹅卵石样细胞层,传代后的细胞能摄取DiI-Ac-LDL,免疫荧光显示vWF和CD133 ;pEGFP-HTK重组质粒在EPCs中可以稳定表达,表达的融合蛋白具有HTK和EGFP的双重活性。结论HTK基因修饰兔外周血EPCs对于血管病的治疗可能具有良好的前景。     

红景天苷对内皮祖细胞的辐射防护作用

 目的: 探讨红景天苷对内皮祖细胞(EPCs)的辐射防护作用,并分析其机制。方法: 体外培养正常人外周血EPCs,观察红景天苷对辐射后EPCs活性、黏附、迁移及凋亡的影响。应用CCK-8法检测细胞活性,流式细胞术检测细胞凋亡率, Western blotting法检测细胞内磷脂酰肌醇3-激酶(PI3K)/Akt通路中Akt蛋白表达水平。结果: 4 Gy [⁶⁰Co]γ射线辐射可导致EPCs损伤,不仅细胞活性、黏附和迁移能力下降,而且细胞凋亡增加;与辐射对照组比较, 红景天苷则能部分逆转辐射对EPCs的损伤,提高EPCs的活性、黏附和迁移能力,减少辐射导致的细胞凋亡,增加细胞内磷酸化Akt蛋白的水平。但这些作用可被PI3K特异性抑制剂LY294002削弱。结论: 红景天苷对内皮祖细胞辐射损伤具有一定的防护作用,其机制可能与增强PI3K/Akt通路的作用有关。     

促红细胞生成素促进内皮祖细胞增殖的体外研究及机制探讨

目的：研究人重组促红细胞生成素（rhEPO）对人内皮祖细胞（EPCs）数量和功能的影响并探讨其作用机制。方法: 以不同浓度rhEPO分别作用于15例处于肾衰竭期的患者以及15例健康志愿者EPCs，MTT法检测其增殖，Annexin-V法检测其凋亡，Western blotting法检测Akt蛋白激酶。结果: 100 U/L、600 U/L及1 200 U/L rhEPO均能提高实验组和对照组EPCs的数量及增殖能力，呈剂量依赖性，实验组EPCs数量及功能均显著低于对照组。1 200 U/L rhEPO能显著降低EPCs的凋亡率，提高 Akt蛋白激酶的表达。加入Wortmannin后能阻断这一效应。结论: rhEPO可以显著提高健康人群和肾衰竭患者EPCs的数量与功能，且这一效应与PI3K/Akt信号通路有关。     

Expression of leukocyte-endothelial cell adhesion molecules on monocyte adhesion to human endothelial cells on plasma treated PET and PTFE in vitro

We used a coculture model to evaluate the inflammatory potential of ammonia gas plasma modified PET and PTFE by flow cytometry and immunohistochemistry. In these studies, human endothelial cells from umbilical cord (HUVEC) and promonocytic U937 cells were used. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-) were used as controls. U937 adhesion to endothelium on each surface was evaluated at day 1 and day 7. To further investigate the role of leukocyte–endothelial cell adhesion molecules (CAMs) in cell-to-cell interaction on material surfaces, the expression of the leukocyte–endothelial CAMs: ICAM-1, VCAM-1, PECAM-1, and E-selectin on HUVECs were evaluated after U937 cell adhesion. The results demonstrated that plasma treated PET (T-PET) and treated PTFE (T-PTFE) did not increase U937 cell adhesion compared to the negative control. Maximal adhesion of U937 cells to HUVEC was observed on TNF- stimulated endothelium with significant differences between day 1 and day 7, which is consistent with our prior observation that T-PET and T-PTFE did not cause HUVECs to increase the expression of adhesion molecules. After U937 cell adhesion, the expression of ICAM-1 and VCAM-1 of HUVECs were not different on T-PET and T-PTFE compared with the negative control. However, the expression of E-selectin was reduced on day 1, but not on day 7. The effects of plasma treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, the cell number increased significantly on the surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion and expression of leukocyte–endothelial CAMs. The monocyte adhesion to endothelial cells on surfaces can be used as a tool for the evaluation of material surface modification and further to study the mechanisms of cell-to-cell interactions in response to surfaces.     

Magnetic-based multi-layer microparticles for endothelial progenitor cell isolation,enrichment, and detachment

Although endothelial progenitor cells (EPCs) are useful in many applications including cell-based therapies, their use is still limited due to issues associated with cell culture techniques like a low isolation efficiency, use of harmful proteolytic enzymes in cell cultures, and difficulty in ex vivo expansion. Here, we report a tool to simultaneously isolate, enrich, and detach EPCs without the use of harmful chemicals. In particular, we developed magnetic-based multi-layer microparticles (MLMPs) that (1) magnetically isolate EPCs via anti-CD34 antibodies to avoid the use of Ficoll and harsh shear forces; (2) provide a 3D surface for cell attachment and growth; (3) produce sequential releases of growth factors (GFs) to enrich ex vivo expansion of cells; and (4) detach cells without using trypsin. MLMPs were successful in isolating EPCs from a cell suspension and provided a sequential release of GFs for EPC proliferation and differentiation. The cell enrichment profiles indicated steady cell growth on MLMPs in comparison to commercial Cytodex3 microbeads. Further, the cells were detached from MLMPs by lowering the temperature below 32 °C. Results indicate that the MLMPs have potential to be an effective tool towards efficient cell isolation, fast expansion, and non-chemical detachment.     

血管内皮生长因子对外周血内皮祖细胞功能的影响

目的: 观察不同浓度血管内皮生长因子(VEGF）对体外培养人外周血内皮祖细胞（EPCs）生物学功能的影响，探讨VEGF促进EPCs分裂生长的合适浓度。方法: 密度梯度离心法获取人外周血单个核细胞（MNCs），接种至人纤维连接蛋白（HFN）包被的培养板上，培养4 d后收集贴壁细胞，换用配有不同浓度（对照组、10 μg/L、20 μg/L、50 μg/L）VEGF的培养基继续培养3 d后进行细胞免疫组化鉴定EPCs，采用MTT比色法、改良的Boyden小室和黏附能力测定实验，观察EPCs的增殖、迁移和黏附能力。结果: 从人外周血能成功分离EPCs细胞，并能分化为血管内皮细胞；从冠心病患者分离的EPCs增殖能力较非冠心病患者弱；VEGF在较低浓度（10 μg/L和20 μg/L）时即能显著促进EPCs生长的各项生物学指标，但高浓度（50 μg/L）时并不能进一步增强这一效应；低浓度（10 μg/L）下VEGF对冠心病患者作用较非冠心病患者弱，高浓度时对两者促进作用相近。结论: 冠心病患者EPCs功能显著减弱，较低浓度的VEGF即可显著增强EPCs的各项生物学功能，可能对损伤血管的再内皮化有益。     

人外周血来源内皮祖细胞诱导分化为心肌样细胞的实验研究及机制探讨

目的： 研究人外周血来源内皮祖细胞（EPCs）在体外定向分化为心肌样细胞的能力及其可能机制。方法： 分离培养及鉴定健康人群（对照组）及冠心病（CAD）患者（实验组）EPCs。将对照组及实验组EPCs分别分成3组在体外进行分化诱导实验：A组 EPCs与乳鼠心肌共培养；B组 EPCs与多聚甲醛固定后的乳鼠心肌共培养；C组 于EPCs中加入收集到的乳鼠心肌培养液上清诱导培养。观察心肌样细胞形态变化;2周后免疫荧光法检测心肌肌钙蛋白 （cTnT）和结蛋白（desmin）的表达；逆转录-聚合酶链反应（RT-PCR）检测心肌相关基因ANP、cTnT、cTnI和主要组织相容性复合物α链（αMHC）的表达；流式细胞仪检测HLA-DR和人desmin的表达，计数desmin阳性表达细胞。结果： 实验及对照A、B组EPCs均可呈现心肌样细胞形态;ANP、cTnT、cTnI和αMHC基因及cTnT和desmin蛋白均呈阳性表达；流式检测HLA-DR及desmin呈双阳性表达，对照A组分化率为（9±2）％，实验A组分化率约为（4±1）％，差异显著（P<0.01）。实验及对照C组EPCs形态未发生变化，各项检测指标均为阴性表达。结论： 与乳鼠心肌共培养可诱导人外周血来源EPCs分化为心肌样细胞，且其可能机制为细胞间的直接接触。     

Involvement of bone marrow-derived endothelial progenitor cells in glomerular capillary repair in habu snake venom-induced glomerulonephritis

Neovasculogenesis is essential in tissue remodeling. Endothelial progenitor cells (EPCs) mobilize from bone marrow (BM) and participate in neovasculogenesis. This study examined the role of EPCs in a model of reversible glomerulonephritis induced by habu snake venom (HSV). Lethally irradiated FVB/N wild-type mice were transplanted with BM cells from donor transgenic mice expressing beta-galactosidase gene under the control of endothelial-specific tie-2 promoter. HSV or saline was injected intravenously after BM transplantation (BMT). The kidneys were removed before injection and at days 1, 7, 28, and 56 after injection. beta-Galactosidase-expressing cells were identified by X-gal staining. The expressions of CD31 (endothelial cell marker) and vascular endothelial cell growth factor (VEGF) in renal tissues were examined by immunohistochemistry. In BMT mice injected with saline, few X-gal-positive cells were detected in glomeruli. In HSV-injected mice, X-gal-positive EPCs were increased in damaged glomeruli, reaching maximum at day 28. Recovery of glomeruli was observed at day 56 in association with reduction of X-gal-positive EPCs. VEGF overexpression was detected in glomerular epithelial and endothelial cells, mesangial cells, and EPCs. Our results indicated that EPCs were mobilized into the damaged glomeruli, suggesting EPCs participation in glomerular capillary repair of damaged glomeruli in HSV-induced glomerulonephritis.     

Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence

Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.     

Paracrine mechanisms of endothelial progenitor cells in vascular repair

Endothelial progenitor cells (EPCs) play an important role in repairing damaged blood vessels and promoting neovascularization. However, the specific mechanism of EPCs promoting vascular repair is still unclear. Currently, there are two different views on the repair of damaged vessels by EPCs, one is that EPCs can directly differentiate into endothelial cells (ECs) and integrate into injured vessels, the other is that EPCs act on cells and blood vessels by releasing paracrine substances. But more evidence now supports the latter. Therefore, the paracrine mechanisms of EPCs are worth further study. This review describes the substances secreted by EPCs, some applications based on paracrine effects of EPCs, and the studies of paracrine mechanisms in cardiovascular diseases--all of these are to support the view that EPCs repair blood vessels through paracrine effects rather than integrating directly into damaged vessels.     

同型半胱氨酸影响内皮祖细胞机制的探讨

目的：探讨同型半胱氨酸（Hcy）影响内皮祖细胞（EPCs）数量和功能的可能机制。 方法：采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的培养板,培养4 d后,收集贴壁细胞,加入不同浓度Hcy（10 μmol/L、30 μmol/L、100 μmol/L和200 μmol/L）干预,或先予以1 μmol/L 阿托伐他汀预处理1 h,然后再用200 μmol/L Hcy干预。采用SA-β-半乳糖苷酶染色试剂盒检测衰老细胞,细胞增殖ELISA试剂盒和集落生成能力测定实验检测EPCs的增殖能力和集落形成能力,端粒重复序列扩增法(TRAP)-ELISA定量检测端粒酶活性,Western blotting检测EPCs Akt Ser473磷酸化水平。结果：Hcy呈浓度依赖性增加SA-β-半乳糖苷酶阳性细胞数量,200 μmol/L最为显著,较对照增加了2倍（15.2±9.8 vs 51.9±13.5,P<0.01）,而阿托伐他汀能显著减少Hcy诱导的衰老细胞的数量。此外,Hcy干预后伴随EPCs增殖和集落形成能力的显著损害。Hcy随着浓度的增加而显著降低EPCs端粒酶活性。进一步研究发现Hcy显著减少Akt磷酸化。结论：Hcy加速EPCs衰老,伴随EPCs增殖和集落形成能力的损害,提示细胞衰老也许是Hcy损害EPCs的机制之一。Hcy加速EPCs衰老可能跟EPCs端粒酶活性下降以及Akt磷酸化水平的下降有关。阿托伐他汀可以预防Hcy对EPCs的损害效应。