鼠外周血内皮祖细胞体外培养鉴定与诱导分化

目的探讨鼠外周血来源的血管内皮祖细胞（EPCs）体外培养鉴定及诱导分化的方法。方法从鼠外周血中分离获取EPCs,进行体外培养扩增,并在培养液中添加VEGF及bFGF,采用免疫组织化学技术和流式细胞仪进行EPCs的细胞表面标志物检测。结果培养3-4d,可观察到梭形贴壁细胞,14d左右贴壁细胞呈条索状结构,贴壁细胞是表达血管内皮细胞的特异性标志。结论鼠外周血中含有EPCs,在一定条件下,可分化成血管内皮细胞。     

大鼠骨髓来源的血管内皮祖细胞的体外培养和鉴定

目的探索大鼠骨髓来源的血管内皮祖细胞（EPCs）的体外培养、诱导分化及鉴定方法。方法采用密度梯度离心法从Wistar大鼠骨髓中分离出骨髓单个核细胞,并在诱导培养基（EGM-2MV）中培养,贴壁筛选法分离,诱导分化为EPCs;观察EPCs的生长分化过程,对其形态、表型、功能加以鉴定。结果培养3 d,细胞贴壁较为完全,第4天换液后,细胞呈现克隆样生长,第7～8天形成类似成熟血管内皮细胞形态,呈现典型的"铺路石"样外观;诱导培养第7、10天的贴壁细胞CD34、Flk-1及CDl33染色均呈阳性;荧光显微镜下观察,DIL-ac-LDL和FITC-UEA-1双荧光染色的细胞数占贴壁细胞数的75%以上。结论采用密度梯度离心法获得骨髓单个核细胞,在培养液中贴壁培养,可以扩增出具有典型特征的EPCs。     

人外周血内皮祖细胞培养及特性

目的 探讨人外周血来源的内皮祖细胞(EPCs)体外诱导培养和扩增的方法，以期证实人外周血是临床治疗缺血性疾病一个理想的内皮祖细胞来源。方法 采集正常人外周血单个核细胞进行体外培养，应用免疫组织化学和免疫荧光技术进行内皮细胞表面标记物检测，应用细胞荧光化学检测进行培养细胞的内皮细胞功能检测。结果 人外周血单个核细胞(MNCs)经体外培养，表达内皮细胞的特异性抗原，包括Ⅷ因子(VWF)、FLK-1、CD31和CD34，并显示出能内吞乙酰低密度脂蛋白(acLDL)，结合UEA-1等内皮细胞的特性，提示这些培养细胞具有内皮细胞的表面蛋白特性和具有内皮细胞的功能。结论 人外周血中存在具有分化增殖能力的EPCs，可体外扩增和收集。     

外周血早晚期内皮祖细胞的生物学特性及鉴定的初步研究

目的通过人外周血分离培养早晚期内皮祖细胞(endothelial progenitor cells,EPCs),比较两类EPCs的特性,以期找到可靠的稳定生物学表征及鉴定方法鉴定EPCs。方法通过密度梯度离心法分离人外周血单核细胞,体外种植于内皮细胞全培养基诱导培养,分别于培养4~7 d及2~3周获得早、晚期EPCs。对两类EPCs的细胞形态、增殖能力、细胞表型、细胞因子表达情况、体外成血管能力及一氧化氮(nitric oxide,NO)释放能力进行检测比较,并以成熟人主动脉内皮细胞(human aortic endothelial cells,HAECs)作为阳性对照。结果两类EPCs形态不同,早期EPCs形成纺锤样细胞簇,而晚期EPCs呈鹅卵石样外观;晚期EPCs具有高增殖潜能,可在基质胶上形成毛细管状结构,而早期EPCs则不具备该特性;两类EPCs均可摄取乙酰化低密度脂蛋白,并能与荆豆凝集素Ⅰ结合;流式细胞仪检测发现早期EPCs不表达CD34和CD133,但造血干细胞标记CD14和CD45表达却呈阳性,而晚期EPCs对于内皮型表面标记CD31和CD34表现为强阳性,但CD14、CD45、CD133表达则呈阴性。RTPCR分析发现早期EPCs的血管内皮生长因子受体2及血管内皮钙黏蛋白基因的相对表达量显著低于晚期EPCs及HAECs(P0.05),而血管性血友病因子及内皮型一氧化氮合成酶(endothelial nitric oxide synthase,eNOS)基因的相对表达量比较差异无统计学意义(P0.05)。细胞因子分泌方面,早期EPCs培养上清液中的VEGF、粒细胞集落刺激因子及IL-8浓度明显高于晚期EPCs(P0.05)。Western blot检测示,两类EPCs均表达eNOS,但培养5周的晚期EPCs的eNOS表达水平高于早期EPCs(P0.05);两种EPCs均可释放NO,但NO产量差异无统计学意义(P0.05)。结论 eNOS的表达及NO释放能力作为内皮细胞的可靠生物学特征可用于EPCs鉴别,多方法联合方式用于鉴定EPCs更为可行。     

大鼠脐血来源单个核细胞诱导为内皮祖细胞的免疫荧光检测

目的脐血（umbilical cord blood,UCB）中含有大量内皮祖细胞（endothelial progenitor cells,EPCs）,内皮祖细胞（EPCs）是血管内皮细胞的前体细胞,有分化为血管内皮细胞的能力,为骨组织工程血管化带来新的途径。本实验研究大鼠脐血来源的单个核细胞体外诱导为EPCs的可能性。方法分离大鼠脐血单个核细胞,血管内皮诱导液（添加10％胎牛血清,1％青霉素,1％链霉素,VEGF20ug／L,IGF-12ug／L,bFGF2ug／L,EGF20ug／L）培养分化,于3周、6周进行免疫荧光染色检测贴壁细胞CD34、CD133、Flk-1、vWF表面标志。结果培养三周单个核细胞CD34、CD133、Flk-1、vWF抗体免疫荧光检测均为阳性;六周免疫荧光染色可见CD34、Flk-1、VWF组为阳性,CD133组部分为阳性,阴性对照组未发现阳性细胞。结论大鼠脐血来源的单个核细胞在体外诱导3周可以分化为EPCs,6周部分细胞分化为成熟血管内皮细胞。     

血管内皮细胞生长因子基因转染血管内皮祖细胞移植提高移植脂肪存活率的实验研究

目的探讨血管内皮细胞生长因子165(VEGF165)基因转染血管内皮祖细胞(EPCs)移植促进游离移植的脂肪组织的血管新生,提高移植脂肪组织存活率。方法体外分离、培养人脐血中EPCs,利用脂质体介导VEGF165基因体外转染EPCs,然后与来自人体的脂肪组织混合移植于裸鼠背部,裸鼠随机分为3组:VEGF165基因转染组、EPCs组及M199培养基对照组。结果脐血中分离培养的EPCs表达CD34、血管内皮细胞生长因子受体及CD133;VEGF165基因转染EPCs体外及体内检测均有VEGF165蛋白的表达。VEGF165基因转染组、EPCs组中,EPCs整合到缺血部位新生血管中,与对照组的脂肪组织存活率分别为(96.2±8.6)%、(75.3±6.8)%和(40.2±2.5)%(P<0.05),VEGF165基因转染组与EPCs组脂肪组织周边区毛细血管密度有显著差异(P<0.05),均高于对照组(P<0.05)。术后3个月时3组脂肪组织周边区的EPCs密度分别为(196±16)个/mm2、(95±11)个/mm2、0个/mm2(P<0.05)。结论脐血中的EPCs体外培养后移植体内可促进游离移植的脂肪组织的血管新生,提高存活率,而转染VEGF165基因的EPCs具有更强的促血管新生的作用。     

人骨髓内皮祖细胞的分离、诱导培养和扩增

目的 探讨并改进从人骨髓中分离、诱导培养和体外扩增内皮祖细胞(EPCs)的方法 ,为EPCs参与基础研究和临床应用奠定基础. 方法 采用不同的细胞分离液,用密度梯度离心法从正常人骨髓中分离单个核细胞(hBMMNCs),分别培养在包被人纤维连接蛋白(HFN包被组),包被明胶(明胶包被组)和未包被(未包被组)的培养皿内,利用EBM-2培养4～7 d后出现细胞克隆集落(cell colony-forming units,CFUs),挑选内皮祖细胞样CFUs继续培养(CFUs挑选法),采用流式细胞仪检测CD34 KDR 和CDl33 KDR 双荧光阳性细胞,细胞免疫化学法检测EPCs表面标记CD133、CD34、CD31、vWF和KDR的表达. 结果 用OptiprepTM分离液可从人骨髓中更有效地分离出hBMMNCs,进而诱导获取更多EPCs并可进行体外培养扩增;流式细胞仪检测结果 显示CD133 KDR 细胞高达70.4%±5.4%,CD34 KDR 细胞高达69.1%±8.7%;HFN包被组和明胶包被组细胞生长一样旺盛,均可促进EPCs的贴壁生长,促进其生长的作用差异无统计学意义(P＞0.05),而未包被组细胞生长数量最少.培养7d的贴壁细胞表面标记CD133、CD31、vWF和KDR均呈阳性,培养14d的贴壁细胞表面标记CD34呈阳性. 结论 CFUs挑选法可以从人骨髓中成功地获取更多EPCs并在体外扩增,提出了另一种获取EPCs的高效简单方法 ,进一步拓宽了获取EPCs的方法 和范围.     

兔外周血内皮祖细胞的分离培养及鉴定

内皮细胞(EC)构成正常的血管壁,内皮祖细胞(EPCs)是EC的前体细胞,可在体内、外分化为成熟EC。本研究以新西兰兔外周血为研究对象,探索分离、培养、扩增EPCs的方法,以期为细胞治疗为主的血管性疾病治疗提供实验基础。     

人VEGF165基因转染人外周血内皮祖细胞的实验研究

目的 探讨人血管内皮细胞生长因子165(hVEGF165)基因转染对人外周血内皮祖细胞(EPCs)的影响.方法 体外分离、培养、鉴定人外周血EPCs.实验分脂质体介导pcDNA3.1-hVEGF165质粒转染组,pcDNA3.1空质粒转染组,窄白对照组.ELISA法和硝酸还原酶法分别测定各组上清液中VEGF和一氧化氮(NO)的含量;噻唑蓝(MTT)检测它们对EPCs增殖的影响.结果 FITC-UEA-I和DiI-ac-LDL双染色阳性细胞为正在分化的EPCs,脂质体介导pcDNA3.1-hVEGF165质粒转染组EPCs培养基上清液中VEGF和NO表达量明显高于其他两组(P＜0.01);VEGF质粒转染对EPCs的增殖无明显影响.结论 人外周血EPCs可以成功转染hVEGF165基因.同时能表达一定浓度的VEGF蛋白,并可促进NO的分泌,而对EPCs的活性无明显影响.该研究为进一步研究VEGF165基因和EPCs结合治疗缺血性疾病提供了实验依据.     

内皮祖细胞在肾癌中的研究进展

内皮祖细胞(Endothelial progenitor cells,EPCs)主要来源于骨髓,是内皮细胞的前体细胞,多种因素可动员EPCs入外周血参与血管新生.因此,EPCs有可能成为一种抑制肿瘤血管生成的靶向载体,在肾癌治疗中正引起人们的关注.本文就EPCs在肾癌中的研究进展进行了综述.     

Differentiation and proliferation of endothelial progenitor cells from canine peripheral blood mononuclear cells

BACKGROUND: The isolation, differentiation, and expansion of endothelial progenitor cells (EPCs) from peripheral blood have potential applicability in areas of therapeutic neovascularization, vascular repair, and tissue engineering. The purpose of the current study was to elucidate a simple method of isolation and differentiation of EPCs by defining the endothelial morphology, surface marker expression, and proliferative capacity of EPC outgrowth from canine peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: PBMCs were isolated from fresh canine blood and cultured in fibronectin-coated plates in which EPCs were identified from cell morphology and outgrowth characteristics. Cell surface markers were determined with flow cytometry analysis to identify differentiation of cultured and subcultured colonies. A hematologic counter with phase contrast microscopy was used to study cell growth curves of EPCs as compared with mature human coronary artery endothelial cells. RESULTS: During the first week of canine PBMC culture, cells were morphologically round and varied in size, but in the course of the second and third week of culture, the cells, respectively, became spindle-shaped and displayed an endothelium-like cobblestone morphology with outgrowth. CD34 was significantly decreased at 21 days as compared with 7 days culture (36.04% to 21.37%), whereas vWF (from 77.26% to 96.37%) and eNOS (from 0% to 14.97%) were significantly increased. VEGFR-2 was slightly increased, and P1H12 (CD146) was unchanged. Subcultured canine EPCs displayed a higher proliferation rate as compared to mature human coronary artery endothelial cells in the same culture conditions. CONCLUSIONS: These data demonstrate that canine EPCs can be isolated and cultured from the canine PBMC fraction. These outgrowth cells displayed characteristics of endothelial morphology with endothelial cell-specific surface markers. Furthermore, it was revealed that canine EPCs have a greater growth potential as compared to mature endothelial cells. This study suggests that PBMCs could be used as a source of EPCs for potential applications in tissue engineering and vascular therapy.     

血管内皮生长因子和碱性成纤维细胞生长因子对大鼠外周血内皮祖细胞培养的影响

目的 探讨不同培养条件对大鼠外周血来源内皮祖细胞（EPC）生长情况的影响。 方法 密度梯度离心法获得大鼠外周血单个核细胞,根据培养基中是否添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）及培养板是否预铺纤连蛋白（FN）分组培养。观察记录细胞生长情况并进行统计分析,以免疫组化和免疫荧光法进行鉴定。 结果 大鼠外周血来源的单个核细胞在体外呈现贴壁生长,培养第7天各组细胞数及细胞集落数提示,在相同培养条件下,预铺FN可以促进EPC的贴壁增殖（t = 4.43,P < 0.05;t = 3.70,P < 0.05）。在同样预铺或未铺FN的情况下,在培养液中加入生长因子可促使单个核细胞更好地向EPC分化（t = －10.96,P < 0.01;t = －13.22,P < 0.01）。免疫组化及免疫荧光结果显示,细胞培养第4、7、10天,细胞表面CD34、CD133表达不断增强[（35.7±4.2）%、（60.1±3.8）%、（81.8±6.4）%;（3.2±0.9）%、（18.4±7.3）%、（32±3.8）%],第14天下降[（32.1±5.4）%,（1.9±2.7）%];而Flk-1表达在第4、7、10、14天均不断增强[（31.2±3.5）%、（40.6±5.3）%、（71.2±8.4）%、（81.5±4.1）%]。 结论 FN有利于内皮祖细胞的贴壁生长和增殖。VEGF及bFGF促进其增殖分化。内皮祖细胞的体外成功培养将为其应用于血管组织工程提供足够数量的种子细胞来源,并为外周血干细胞移植治疗多种疾病提供新的思路。     

人脐血CD133+血管内皮祖细胞的培养、鉴定与分化研究

目的探讨从人脐血中分离、体外培养CD133^＋血管内皮祖细胞（EPCs）的方法及其生长特性和鉴定。方法采用密度梯度离心法结合MACS分选法提取人脐血中CD133^＋细胞,进行流式细胞仪检测纯度,予EBM-2培养液接种培养,观察其生长特性;利用Dil-LDL及FITC-Lectin摄取实验、细胞免疫组织化学等进行鉴定。结果经流式细胞仪检测CD133^＋细胞平均占单核细胞的（1．13±0．10）％,磁珠分选所得CD133^＋细胞平均纯度为（91．45±1．04）％;CD133^＋细胞贴壁生长,可分化为梭形血管内皮细胞及形成集落;CD133^＋细胞培养过程中Dil-LDL及FITC-Lectin摄取阳性,双染阳性率为（95．83±1．72）％;CD133^＋细胞培养1周后经免疫组织化学检测CD34、Ⅷ因子阳性率分别为（95．83±2．23）％和（95．92±1．43）％,与人脐静脉内皮细胞比较差异无统计学意义;CD133^＋细胞体外培养4d、1周可形成小血管结构。结论免疫磁珠分选法可获取较高纯度CD133^＋血管内皮祖细胞,在生长因子作用下诱导其分化为成熟血管内皮细胞。     

Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps

BACKGROUND: Neovascularization occurs through two mechanisms: angiogenesis and vasculogenesis. Therefore, there are two strategies to promote neovascularization: therapeutic angiogenesis and therapeutic vasculogenesis (endothelial progenitor cells therapy). MATERIALS AND METHODS: In this study, we examined whether or not endothelial progenitor cells combined with vascular endothelial growth factor (VEGF) gene therapy is useful for ischemia surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted endothelial progenitor cells (EPCs) transducted with VEGF165 gene and EPCs alone. EPCs were isolated from cord blood of healthy human volunteers, cultured in vitro for 7 days and identified by immunofluorescence. After transduced with VEGF165 gene in vitro, proliferative activity of EPCs was assessed using MTT assay. CM-DiI was used to trace EPCs in vivo 4 days after injection of 5 x 10(5) VEGF-transduced EPCs(VEGF-transduced EPCs group, n = 10), 5 x 10(5) EPCs (non-transduced EPCs group, n = 10) in 500 microL EBM-2 media, or 500 microL EBM-2 media (EBM-2 media group, n = 10) local, a cranially based flap was elevated on the back of nude mice. The percent flap survival, neovasculariztion and blood flow recovery of flaps was detected. RESULTS: EPCs expressed cell markers CD34, KDR, and CD133. A statistically significant increase in percent flap survival was observed in mice of VEGF-transduced EPCs group as compared with that of non-transduced EPCs group: 67.99 +/- 6.64% versus 59.43 +/- 4.69% (P < 0.01), and 41.24 +/- 2.44% in EBM-2 media group (P < 0.01). The capillary density and blood flow recovery of flaps in VEGF-transduced EPCs group were both improved. CM-DiI-labeled VEGF-transduced EPCs were observed in vivo and the numbers of cells increased. CONCLUSION: EPCs from human cord blood can increased neovascularization of ischemic flaps and augmented the survival areas, and VEGF-transduced EPCs have more powerful ability of promoting neovascularization in animal model of ischemic flaps.     

Transplanted endothelial progenitor cells augment the survival areas of rat dorsal flaps

Endothelial progenitor cells (EPCs) have been identified in peripheral blood, and have been reported to be incorporated into ischemic regions such as the ischemic hindlimb. In this study, we examined whether or not transplantation of EPCs is useful for salvaging surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted EPCs and that of mature endothelial cells (ECs). ECs obtained from the aorta of rats by explantation and passaged several times were used in the present study. EPCs were obtained from the blood of rat hearts. The blood samples were separated by density gradient centrifugation. Light-density mononuclear cells (MNCs) were collected and cultured on plastic plates coated with rat plasma vitronectin. Cells attached at day 7 of culture were deemed to be EPCs. Then PBS (control), ECs, or EPCs (3.0 x 10(5) suspended in 1.0 ml PBS) were injected at the middle of a flap. Seven days after surgery, the survival lengths of the flaps were evaluated. EPC-transplanted groups revealed statistically significant augmentation of survival length compared with the other two groups (p < 0.003). EPC-transplanted groups had significantly more angiographically detectable blood vessels (p < 0.003) and significantly higher capillary density (p < 0.03) than the other two groups. Confocal microscopy revealed that EPCs were incorporated into enhanced neovascularization. These results suggest that transplantation of EPCs may be useful for salvaging surgical flaps, and EPCs are superior to ECs in neovascularization ability.     

VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease

BACKGROUND: Direct transfection of ischemic myocardium with naked plasmid DNA encoding for vascular endothelial growth factor-165 (VEGF165) has been shown to mobilize endothelial progenitor cells (EPCs). This study examined the kinetics of circulating EPCs isolated from peripheral blood mononuclear cells after gene transfer, and their role in neovascularization of ischemic myocardium. METHODS: The mononuclear cell population was isolated from peripheral venous blood samples of patients with functional class III or IV angina receiving intramyocardial VEGF165 gene transfer. Peripheral blood mononuclear cells were examined by an in vitro EPC culture assay and fluorescent-activated cell sorting. The data were compared with a control group consisting of patients who had undergone off-pump coronary artery bypass grafting without receiving gene transfer. RESULTS: Coinciding with a rise in VEGF levels, mobilization of EPCs increased significantly over base line for 9 weeks after the treatment (121+/-14 cells/mm2 versus 36.8+/-8 cells/mm2, p < 0.0005), followed by a subsequent decrease. Fluorescent-activated cell sorting analysis confirmed culture assay data, with a statistically significant rise in cells expressing vascular endothelial-cadherin, CD51/61 [alphavbeta3], CD62E [E-selectin], CD34, and KDR. The control group failed to show significant mobilization of EPCs. CONCLUSIONS: Mobilization of EPCs with resultant postnatal vasculogenesis, may play a role in revascularizing ischemic myocardium following human gene transfer with VEGF165.     

骨髓血管内皮祖细胞移植提高缺血皮瓣存活率的研究

目的：探讨大鼠骨髓来源的血管内皮祖细胞（endothelial progenitor cells,EPCs）移植促进缺血皮瓣的血管新生,从而提高皮瓣存活率的可能性。方法：在Wistar大鼠背部形成2cm×8cm蒂部位于双侧髂棘连线上的随意型超比例皮瓣,建立缺血皮瓣模型。采用密度梯度离心法从Wistar大鼠骨髓中分离出骨髓单个核细胞,并在专有的诱导培养基（EG-M2MV）中培养,贴壁筛选法分离,诱导分化为EPCs;对细胞的形态、表型、功能加以鉴定。将EPCs注射移植于iWstar大鼠背部的缺血皮瓣,大体观察皮瓣的存活率,vWF免疫酶染色法检测皮瓣毛细血管密度。以缺血皮瓣单纯注射磷酸盐缓冲液（PBS）的Wistar大鼠作为对照组。结果：骨髓中分离培养的EPCs呈现典型的＂铺路石＂样外观,表达CD34、Flk-1及CDl 33表型,能够摄取DIL-ac-LDL和特异性结合FITC-UEA-1。注射EPCs组的缺血皮瓣存活率以及毛细血管密度显著高于对照组（P〈0.05）。结论：骨髓中的EPCs在体外培养后注射移植于皮瓣内,可以促进缺血性皮瓣的血管新生,提高缺血性皮瓣的存活率。     

端粒酶反转录酶在体外培养的内皮祖细胞中的表达

目的 探讨体外培养的大鼠内皮祖细胞增殖活性与端粒酶反转录酶表达的关系。 方法 原代培养大鼠骨髓源性内皮祖细胞。在培养7、14、21和28 d时,用MTT法检测内皮祖细胞在不同时间点的增殖活性;流式细胞术检测内皮祖细胞早期凋亡率;免疫细胞化学方法、RT-PCR和Western印迹技术检测内皮祖细胞端粒酶反转录酶mRNA和蛋白表达的变化。 结果 大鼠骨髓单个核细胞可以被诱导成内皮祖细胞。内皮祖细胞增殖活性随着培养时间的延长呈现单峰曲线,在培养14 d时增殖活性最高（P < 0.01）。内皮祖细胞凋亡率随培养时间的延长而增加,在培养7 d、14 d、21 d和28 d时分别为0.28%、0.66%、1.38%、1.52%;衰老率分别为3.04%、20.28%、24.36%、16.52%,14 d 内皮祖细胞衰老率显著增加 （P < 0.01）,21 d衰老率最高,但二者之间差异无统计学意义。端粒酶反转录酶mRNA和蛋白表达呈单峰曲线,在培养14 d时表达最高,以后表达随培养时间的延长逐渐降低（P < 0.05）。 结论 大鼠骨髓内皮祖细胞增殖活性下降可能与端粒酶反转录酶活性下降有关。