内皮祖细胞与氧化应激研究进展

背景：内皮祖细胞是外周血中存在的一种干/祖细胞,是成熟内皮细胞的前体细胞,其与氧化应激的关系越来越受到人们的关注,而且国内外研究正日趋增多。 目的：对国内外内皮祖细胞与氧化应激关系的现状及新进展作一综述。 方法：应用计算机检索CNKI和Pubmed数据库中1997-01/2010-05关于内皮祖细胞与氧化应激的文章,在标题和摘要中以“内皮祖细胞,氧化应激”或“endothelial progenitor cells ,oxidative stress”为检索词进行检索。选择文章内容与内皮祖细胞与氧化应激有关者,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到212篇文献,最终选择有代表性的34篇文献进行综述。 结果与结论：内皮祖细胞与氧化应激关系密切,虽然内皮祖细胞具有比成熟内皮细胞更强的抗氧化能力,但在长期氧化应激存在的情况下,氧化应激通过减弱其抗氧化酶的表达,增加氧化酶的表达而促进内皮祖细胞的凋亡而影响其功能及数量,故氧化应激是导致内皮祖细胞数量改变及功能受损的影响因素之一,而通过他汀类药物、血管紧张素转换酶抑制剂/受体拮抗剂及过氧化物酶体激动剂的干预可改善内皮祖细胞的氧化应激状态,保护其功能。     

白藜芦醇抑制叔丁基过氧化物诱导的外周血内皮祖细胞损伤

目的:观察白藜芦醇对叔丁基过氧化物诱导的人外周血内皮祖细胞(EPC)凋亡的影响,探讨其可能机制。方法:密度梯度离心法获取人外周血单个核细胞,培养4 d后,收集贴壁细胞。实验分为对照组、叔丁基过氧化物诱导组和白藜芦醇+叔丁基过氧化物组。采用二苯基四氮唑溴盐(MTT)比色法和5-溴脱氧尿嘧啶核苷(Brd U)ELISA法检测EPC增殖能力;采用改良Boyden小室法检测EPC增殖及迁移能力;Annexin V-FITC/PI染色及流式细胞术检测EPC凋亡率;比色法检测caspase-3活性;荧光探针H_2DCF-DA法检测细胞内活性氧簇(ROS)水平;Western blot检测cleaved caspase-3、Bax及Bcl-2蛋白的表达。结果:白藜芦醇能抑制叔丁基过氧化物诱导的EPC凋亡,并增加EPC增殖及迁移能力;白藜芦醇降低EPC细胞内ROS水平,抑制caspase-3的活性及cleaved caspase-3的蛋白水平,同时能促进Bcl-2及抑制Bax蛋白表达。结论:白藜芦醇对叔丁基过氧化物诱导的EPC凋亡有抑制作用,其机制可能与抗氧化应激有关。     

前列腺素I2-内皮祖细胞保护氧化应激受损心肌细胞凋亡： 心肌电生理验证

BACKGROUND:Prostacyclin (PGI2) and its analogs have been reported to prevent pressure overload-induced cardiac hypertrophy, and to reduce cardiac ischemia/reperfusion injury. However, clinical application of PGI2 is challenging due to its short half-life (< 2 minutes). Thus, we have generated PGI2 expressing rat endothelial progenitor cell strains (PGI2-EPCs) that constitutively secrete prostacyclin. OBJECTIVE:To investigate the protective effect of PGI2-EPCs against oxidative stress-induced cardiomyocyte injury. METHODS:Cultured H9c2 cells in vitro were assigned into four groups: H9c2 cells treated by H2O2 for 4 hours. H9c2 cells were pretreated by conditioned medium (collected form EPCs and PGI2-EPCs or collected form EPCs and PGI2-EPCs mixed with native EPCs) before the addition of H2O2. PBS instead of conditioned mediums served as negative control. The paracrine effect of PGI2-EPCs on in vitro angiogenesis of native EPCs was evaluated. MTT and Hoechst 33342 assays were used to examine the protective effect of conditioned medium on H2O2-induced rat embryonic cardiomyocyte apoptosis and cell viability. Finally, the effect of conditioned medium on the electric activities of adult cardiomyocytes was measured by whole-cell patch clamp techniques. RESULTS AND CONCLUSION:When native EPCs mixed with conditioned medium of PGI2-EPCs, the total length of tubes was significantly longer compared with those mixed with CM of EPC. Rat embryonic cardiomyocytes pretreated with conditioned medium of PGI2-EPCs significantly reduced H2O2-induced apoptosis and preserved cell viability compared with pretreatment with EPC-conditioned medium and without pretreatment (P < 0.01). Pretreatment of rat adult cardiomyocytes with conditioned medium of PGI2-EPCs abolished H2O2-induced early after depolarization and shortened H2O2-induced action potential duration prolongation (P < 0.01) towards baseline. Our findings indicate that PGI2-EPCs protect against oxidative stress-induced cardiomyocyte injury through paracrine action. This Study provides the groundwork for an innovative cell therapy approach to treat ischemic heart disease. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

α-硫辛酸对高糖状态下内皮祖细胞的影响

目的:应用高浓度葡萄糖孵育大鼠骨髓内皮祖细胞(EPCs),测定培养上清的丙二醛(MDA)、一氧化氮(NO)水平,同时观察EPCs GPx-1、eNOS的表达,然后应用抗氧化剂ALA进行干预,来探讨葡萄糖引发EPCs损伤的机制及治疗措施。方法:清洁级健康雄性Wistar大鼠(体质量180～200g)15只,分离培养EPCs,用2.5 g/L胰酶/0.2 g/LEDTA消化培养4 d的EPCs,分组贴壁24 h后分别给予葡萄糖5 mmol/L作为正常对照组(NC)、30 mmol/L葡萄糖作为高糖组(HS)以及葡萄糖(30 mmol/L)+α硫辛酸(40μg/L)作为ALA组,孵育EPCs 48 h,实时荧光定量PCR技术及Western blot法测定EPCs GPx-1及eNOS的mRNA及蛋白表达,测定培养液上清的一氧化氮(NO)及MDA水平。结果:(1)不同干预措施对EPCs分泌MDA的影响:高浓度葡萄糖干预48 h后培养上清MDA水平高于正常对照组(P<0.05);应用ALA干预后与高糖组比较MDA水平下降(P<0.05)。(2)不同干预措施对EPCs GPx-1表达的影响:高糖干预48 h后EPCs GPx-1表达显著低于对照组,而ALA干预后EPCs GPx-1表达较高糖组显著增加(P<0.05)。(3)不同干预措施对EPCs eNOS表达及分泌NO的影响:高糖干预48 h后EPCs eNOS表达显著低于对照组,培养上清NO水平低正常对照组(P<0.05),而ALA干预后EPCs eNOS表达较高糖组显著增加(P<0.05),培养上清NO水平升高(P<0.05)。结论:高浓度葡萄糖可以诱发EPCs产生氧化应激,减弱EPCs的抗氧化能力及eNOS表达,使EPCs分泌NO减少,α硫辛酸能够改善高糖导致的EPCs抗氧化能力减弱及NO的分泌。     

内皮祖细胞与肿瘤血管新生

血管内皮祖细胞（EPCs）是内皮细胞的前体细胞,特异性表达CD34,CD133和VEGFR-2,具有向血管内皮细胞分化的潜能。EPCs主要位于骨髓和外周血。肿瘤的生长和转移依赖于肿瘤血管新生。肿瘤细胞可合成和释放多种细胞因子,在不同因子的趋化作用下EPCs从骨髓动员至外周血循环,然后迁移和定居到肿瘤组织,经细胞因子诱导分化为成熟内皮细胞,参与肿瘤血管新生。VEGF/VEGFR-2信号途径在EPCs参与的肿瘤血管新生方面起重要作用。     

内皮祖细胞与雌激素

背景：研究发现雌激素对血管内皮具有明显的保护作用,而内皮祖细胞作为内皮细胞的前体细胞参与内皮的修复。 目的：总结内皮祖细胞生物特点及雌激素对内皮祖细胞作用的研究进展。 方法：应用计算机检索PubMed数据库及CNKI数据库,在标题和摘要中以“内皮祖细胞,雌激素”或“Endothelial progenitor cells,estrogen”为检索词进行检索。选择与内皮祖细胞生物学特点及雌激素对其作用研究相关的文献。 结果与结论：内皮祖细胞存在于骨髓和外周血中,是具有增殖、迁移、黏附能力并分化为血管内皮细胞潜能的原始细胞,可作为未来治疗心血管疾病的重要的靶点。雌激素对内皮祖细胞有保护效应,能增强内皮祖细胞增殖、迁移、黏附等生物活性,同时还能延迟内皮祖细胞衰老、拮抗其凋亡。但雌激素影响内皮祖细胞生物活性的具体靶点及机制尚有待进一步研究。     

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。     

内皮祖细胞的研究进展

骨髓含有内皮祖细胞, 能够迁移至外周血并分化为成熟内皮细胞,参与胚胎时期的血管生成、出生后的微血管新生以及肿瘤组织的发生。体内内皮祖细胞数量和活性受多种生理性及病理性因素的的影响。体外扩增后回输体内可以修复受损组织、器官的血管,促进器官功能恢复;抑制其活性,在一定程度上可以抑制肿瘤组织的生长。内皮祖细胞为缺血性疾病以及肿瘤的治疗提供了另一新的靶点。     

血管内皮祖细胞与糖尿病血管病变

内皮祖细胞(EPC)是能直接分化成血管内皮的前体细胞,参与胚胎期及成体的血管生长。糖尿病内皮祖细胞的数量及功能均有改变,使血管阻塞事件具有更高的发病率和致死率。已有实验证实EPC存在并参与了出生后新血管形成。在动物肢体缺血模型中,EPC移植能扩大缺血组织的侧枝血管生长,恢复血流,改善缺血,这为糖尿病血管病变提供了一种可行性治疗策略。     

Endothelial Progenitor Cells Predict Long-Term Mortality in Hemodialysis Patients

Background: The endothelial progenitor cells (EPCs) dysfunction is a critical event in the initiation of atherosclerotic plaque development and the level of circulating EPCs can be considered a biomarker of cardiovascular events. The level and functional change in EPCs has been investigated in hemodialysis patients, but the effect of absolute number of EPCs on risk of death has not yet been explored. We hypothesized that the number of EPCs predicted death from cardiovascular and all-cause mortality in hemodialysis patients.Methods: We evaluate the association between endothelial progenitor cells and clinical outcome in 154 patients on maintenance hemodialysis. The blood sample was drawn at the time of patient enrollment and EPCs were identified by flow cytometry using triple staining for CD34/CD133/KDR.Results: The median duration of follow-up was 4.19 years. There were 79 (51.3%) deaths during the follow-up period, 41 of whom died due to a confirmed cardiovascular cause. The cumulative survival was greater in the high-EPC group than the low-EPC group for all-cause and cardiovascular mortality. Decreased EPCs levels were associated with a significant increase in the risk of cardiovascular and all-cause mortality after adjusting for age, gender, current smokers, diabetes mellitus, and hypertension.Conclusions: The level of circulating EPCs independently predicts the clinical outcome in patients on maintenance hemodialysis. Thus, the EPCs levels may be a useful predictive tool for evaluating the risk of death in maintenance hemodialysis patients.     

Circulating Progenitor and Mature Endothelial Cells in Deep Vein Thrombosis

Introduction: Mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC) have been described in several conditions associated with endothelial injury. Their role in deep vein thrombosis (DVT) has not been previously evaluated. Patients and Methods: In this pilot study we evaluated the time course of CEC and EPC release after vena cava experimental DVT in mice, using the FeCl3 model. We also evaluated their presence in patients with DVT at different phases of the disease (acute and chronic phase). CEC and EPC were evaluated by Flow Cytometry. Results: In mice, both CEC and EPC were increased 24 hours after DVT induction, peaking 48 hours thereafter. After 72 hours, CEC counts decreased sharply, whereas EPC counts decreased less substantially. In DVT patients we observed a significant increase in CEC counts immediately after DVT compared to healthy individuals. Patients with chronic disease also presented a significant elevation of these cell count. In a subgroup of patients for whom serial samples were available, CEC counts decreased significantly after 9-15 months of the acute event. Conclusions: Our results suggest the participation of these cells in the reparative processes that follows DVT, both at immediate and late time-points. The different kinetics of CEC and EPC release in experimental DVT suggests a heterogeneous role for these cells in the reparative events after DVT.     

内皮祖细胞作为组织工程瓣膜内皮种子细胞的可行性研究

目的 探讨利用外周血内皮祖细胞（EPC8）制备组织工程瓣膜的可行性。方法 分离人外周血EPCs，采用酶-去垢剂法去除新鲜猪主动脉瓣细胞制备去细胞瓣膜支架，将培养的人外周血EPCs接种到去细胞瓣膜上。结果 经酶-去垢剂法去除新鲜猪主动脉瓣细胞后，细胞成分全部去除，纤维支架保存完好。去细胞处理后瓣膜无明显细胞毒性。人外周血EPCs与去细胞瓣膜共孵育2周后，细胞紧贴瓣膜表面生长形成一层连续的单细胞层，初步生物力学测定示去细胞前与再内皮化后，瓣膜力学特性无明显改变。结论 外周血分离培养扩增得到的EPCs能够再内皮化去细胞猪主动脉瓣构建组织工程瓣膜，外周血EPCs是组织工程瓣膜内皮种子细胞的新的来源。     

可注射血管内皮祖细胞-载体材料复合物的体外研究

目的研究可注射性血管内皮祖细胞载体材料复合物的体外生物相容性。方法从犬外周血中分离出单个核细胞体外扩增培养形成贴壁(AT)细胞,将AT细胞分别与藻酸钙(CA)和温度依赖性合成水凝胶(TDSH)溶液混合,混合液中AT细胞的浓度为1×109个/mL,并分别置于相同体系的培养基中体外培养,同时采用相同体系的血管内皮祖细胞作为对照,相差显微镜分别计数培养4、7d的AT细胞数。并分别于第7、29d,采用流式细胞术测定AT细胞中CD34+细胞数和细胞凋亡率。结果第4、7d单个核细胞单独培养和单个核细胞+CA复和培养形成的AT细胞数和细胞簇数量明显多于单个核细胞+TDSH复合培养(P<0.05)。流式细胞术结果显示AT细胞中CD34阳性率在三组无显著性差异。TDSH+单个核细胞组凋亡率明显高于其他两组。结论在体外培养时,藻酸钙与血管内皮祖细胞有良好的生物相容性,可作为血管内皮祖细胞载体材料用于细胞移植。     

人动员外周血成体干/祖细胞定向诱导分化为血管内皮前体细胞的免疫表型分析

观察动员后的人外周血成体干/祖细胞体外定向诱导分化为血管内皮前体细胞（EPC）的免疫表型特征及变化。采用健康成人经粒细胞集落刺激因子（rhG-CSF）动员后的外周血成体干/祖细胞,经贴壁培养法定向分化为EPC,之后通过流式细胞术检测EPC表型。结果显示,培养后的贴壁细胞具有内皮细胞特征,能够与I型荆豆凝集素（UEA-I）结合。流式细胞术检测UEA-I结合率为92.1%,内皮细胞标志物CD31、KDR、CD62E均有不同程度增加,而粒-巨噬细胞集落生成单位（CFU-GM）及CD34表达明显减低,表明诱导分化后细胞的免疫表型发生了相应的变化。     

脐血血管内皮祖细胞的分离和诱导分化

目的 从脐血中分离内皮祖细胞，诱导其向内皮细胞分化，研究内皮祖细胞的生物学特性和诱导分化条件。方法 从新鲜脐血中纯化的CD133^ 细胞接种于添加了VEGF、bFGF、IGF—1的M199培养液中，观察梭形贴壁细胞的出现时间和特异性细胞标志。结果 培养3—4d可观察到梭形贴壁细胞，14d左右可形成索条状结构，贴壁细胞表达血管内皮细胞特异性标志VE-cadherin,vWF,UEA-1和VEGFR-2。结论 脐血中含有内皮祖细胞，在一定的条件下，可分化为内皮样细胞。     

microRNA-214-3p在周期性张应变诱导内皮祖细胞分化和增殖中的作用

目的探讨microRNA-214-3p(miR-214-3p)在周期性张应变诱导内皮祖细胞(endothelial progenitor cells, EPCs)分化和增殖中的作用。方法采用FX-5000T细胞周期性张应变加载装置对EPCs施加生理水平的周期性张应变(5%幅度、1.25 Hz频率),加载时间24 h。应用miRNAs芯片筛选周期性张应变调控下差异表达的miRNAs,并挑选miR-214-3p进行深入研究。实时荧光定量PCR方法检测EPCs内平滑肌细胞(vascular smooth muscle cells, VSMCs)相标志分子的表达,BrdU结合酶联免疫吸附ELISA法检测EPCs增殖功能。之后,使用miR-214-3p抑制剂抑制miR-214-3p的表达,检测EPC内VSMC相标志分子表达及EPCs增殖。结果周期性张应变显著抑制miR-214-3p表达,并抑制EPCs向VSMC相分化,同时显著促进EPCs增殖。在静态条件下,使用miR-214-3p抑制剂干扰miR-214-3p的表达,miR-214-3p水平下降同样会抑制EPCs向VSMC相分化,并且诱导EPCs增殖能力显著上升。结论生理水平的周期性张应变能够抑制EPCs内miR-214-3p表达,从而抑制EPCs向VSMC相分化,并且促进EPCs增殖。研究结果为血管损伤的治疗提供新的治疗靶点。     

Endothelial and Hematopoietic Progenitor Cells (EPCs and HPCs): Hand in Hand Fate Determining Partners for Cancer Cells

Tumor growth and metastasis need new vessel formation by angiogenesis provided by mature endothelial cells and postnatal vasculogenesis provided by endothelial progenitor cells (EPCs). Emerging data suggest a coordinated interaction between EPCs and hematopoietic progenitor cells (HPCs) in these processes. The complexity of the mechanisms governing the new vessel formation by postnatal vasculogenesis has increased by new evidence that not only bone marrow derived EPCs and HPCs seem to be involved in this process but also local progenitors residing within the vascular wall are mobilized and activated to new vessel formation by tumor cells. This review attempts to bring these systemic and local players of postnatal vasculogenesis together and to highlight their role in tumor growth and mestastasis.     

人脐血内皮祖细胞的分离、培养及鉴定

目的探索人脐静脉血内皮祖细胞的分离培养,为内皮祖细胞的临床应用提供实验方法。方法选择脐静脉血,应用密度梯度离心法,获取单个核细胞,接种于预先包埋了人纤维连接蛋白的培养板,用加入生长因子VEGF165和bFGF的内皮细胞专用培养基EGM-2MV培养细胞,3d后,洗掉非贴壁细胞,换培养液继续培养至7d,收集贴壁细胞进行细胞分析。激光共聚焦显微镜进行细胞功能学鉴定,流式细胞术测定祖细胞和内皮细胞系标志。MTT比色法检测细胞的生长状态。结果经过梯度密度离心和贴壁法选择的细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-acLDL,祖细胞标志CD133及内皮细胞特异性抗原CD34、KDR检测,其阳性率分别为（27．05±2．94）％、（16．37±2．69）％和（56．67±7．29）％;体外培养的内皮祖细胞具有良好的细胞增殖活性。结论人脐静脉血中可以分离培养内皮祖细胞,为内皮祖细胞的进一步研究及临床应用奠定了基础。     

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。