小鼠骨髓源性内皮祖细胞的分离培养与鉴定

目的 建立一种高效、稳定的从小鼠骨髓中分离培养与定向诱导分化内皮祖细胞的方法.方法 通过密度梯度离心法从小鼠骨髓中分离单个核细胞,经差速贴壁结合特殊培养基扩增,诱导分化为内皮祖细胞.应用流式细胞技术鉴定内皮细胞系列标志:CD34、CD31、Flk-1和祖细胞标志CD133.结果 经密度梯度离心和差速贴壁法分离所得的细胞经EBM-2专用培养基培养后,第4天可见集落形成,培养第12天流式细胞仪检测其CD34、CD133、Flk-1、CD31的阳性率分别为65%±4%、48%±3%、37%±3%和51%±4%.结论 从小鼠骨髓中分离培养与定向诱导分化内皮祖细胞的方法效率高,稳定性和重复性好.     

骨髓血管内皮祖细胞的分离培养及分化鉴定

目的 研究分离、培养小鼠骨髓血管内皮祖细胞的方法并对其进行诱导分化鉴定,为临床进行缺血性疾病的替代治疗奠定细胞学基础.方法 用灌注法分离小鼠骨髓血管内皮祖细胞,用血管内皮生长因子、成纤维细胞生长因子诱导其分化,形态学观察细胞的生长过程,并利用抗CD34抗体鉴定血管内皮祖细胞,利用抗vWF抗体、抗eNOS抗体鉴定血管内皮细胞. 结果小鼠骨髓血管内皮祖细胞可在体外培养条件下贴壁生长,可诱导分化为表达特异性组织蛋白的内皮细胞.结论 在小鼠骨髓内可分离出骨髓源性血管内皮祖细胞,呈贴壁增殖状态,并有分化能力.     

兔外周血与骨髓源性内皮祖细胞培养鉴定及生物学特性的研究

目的:探索并建立兔内皮祖细胞(EPCs)的体外培养、鉴定方法。方法:采用密度梯度分离法分别从兔外周血和骨髓中分离出单个核细胞,接种于预包被明胶的培养瓶,并应用培养基(EBM-2)诱导培养,诱导分化为EPCs;在倒置显微镜下观察两种源性细胞生长过程;台盼蓝法测定细胞传代成活率;通过绘制生长曲线法、MTT法、DNA周期检测比较两种源性获得的EPCs增殖情况;采用免疫荧光染色观察EPCs相关抗原CD34、CD31、CD133及vWF因子的表达。结果:两种源性EPCs均于培养3～4d完全贴壁,呈克隆样生长,7～9d形成类似成熟血管内皮细胞形态,细胞数量逐渐增多,细胞成活率均为95%。两种源性获得的第2代细胞生长曲线近似"s"形、MTT法显示3～5d细胞增殖较明显,外周血源性EPCs G0-G1期和S+G2+M期所占比例分别为96.48%和3.52%,骨髓源性EPCs G0-G1期和S+G2+M期所占比例分别为97.11%和1.84%。第2代EPCs进行免疫荧光鉴定结果显示:两种源性内皮祖细胞均阳性表达CD34、CD133、vWF因子,CD31弱阳性表达。结论:两种源性的EPCs均可高效获得且在体外稳定培养。与传统的骨髓源性EPCs相比较,从外周血源获得的EPCs是一种可靠、简便的培养方法,为组织工程学提供理想的细胞来源。     

外周血单个核细胞向血管内皮祖细胞分化的体外研究

目的该实验从简化方法、提高得率的角度出发,试图建立直接培养单个核细胞(MNC)而得到血管内皮祖细胞(endothelialprogenitorcells,EPCs)的方法。方法抽取人外周抗凝血,用Ficoll密度梯度离心法分离得到MNC,体外对MNC进行诱导分化培养,然后选择免疫荧光、免疫组化、流式细胞记数和RT-PCR方法鉴定培养所得的细胞是否具有EPCs的特征。结果MNC在培养的第2天开始出现成簇现象;第4天细胞由圆形转变为梭形贴壁细胞,呈条索状或管状分布;第7天85%的贴壁细胞摄取Ac-LDL;第14天免疫荧光和免疫组化检测CD31、CD34、vWF、Flk-1均阳性;同时流式细胞记数检测结果显示CD31和CD34均为阳性;另外RT-PCR结果显示培养的细胞表达内皮细胞特异性基因Flt-1,Flk-1、ecNOS、tie-2等。结论用此方法培养的MNC细胞具备了内皮细胞以及干细胞双重特征,故初步认定,人外周血MNC在体外经适当条件的培养,完全可以分化成EPCs。     

人脐静脉血内皮祖细胞的分离扩增和生物学特征

目的:探索人脐静脉血内皮祖细胞的分离和扩增条件,并观测其生物学特性.方法:采集人脐静脉血,应用密度梯度离心法,分离其中单个核细胞,流式细胞术检测CD133 CD34 阳性率;利用差速贴壁法(48 h内贴壁和48 h后贴壁)联合内皮细胞专用培养基EGM-2培养细胞,接种于预先包埋了明胶培养瓶或培养板,倒置显微镜观察细胞生长形态和形成集落能力,免疫细胞化学法检测其免疫表型,摄取Ac-LDL和连接UEA-1功能,在生长因子培养体系中诱导其向成熟内皮细胞分化.结果:所获单个核细胞中CD133 CD34 百分比为1.06%;在EGM-2培养体系下可获得2种亚型的内皮祖细胞,即早期内皮祖细胞和晚期增殖性内皮祖细胞.其中48h后贴壁细胞属于早期内皮祖细胞,增殖能力较弱,免疫荧光检测,显示CD14和CD34KDR胞浆呈阳性表达,Ac-LDL UEA-1 功能特征;而48 h内贴壁细胞在10～17 d时可见由单个细胞增殖形成的克隆,呈铺路石样单层排列,增殖力旺盛形成融合状态,形成次集集落;经免疫荧光检测,显示CD133CD34和CD34KDR细胞质呈阳性表达,Ae-LDL UEA-1 功能特征,传代后vWF,CD31呈强阳性表达,是晚期增殖性内皮祖细胞.结论:经人脐静脉血可分离培养获得2种亚型的内皮祖细胞,在特定的培养体系中细胞可由祖细胞表型向成熟内皮细胞分化.     

骨髓间充质干细胞体外定向诱导内皮祖细胞的实验研究

目的:研究兔骨髓间充质干细胞(mensenchymal stem cell,MSC)在体外定向诱导分化为内皮祖细胞(endothelial progenitor cell,EPC)的状况。方法:取兔髂骨骨髓,经密度梯度离心法分离并培养骨髓间充质干细胞,实验组细胞在内皮细胞生长添加剂、血管内皮生长因子、成纤维细胞生长因子等诱导条件下定向分化为内皮祖细胞。对照组不干预,继续培养。结果:实验组细胞集落接近融合时形态呈现“鹅卵石”样或“铺路石”样外观,流式细胞仪检测发现实验组细胞较对照组高度表达CD133、CD34(P<0．05)。结论:体外诱导可使骨髓间充质干细胞分化为内皮祖细胞,并有效扩增。     

不同诱导因子下内皮祖细胞具有双向分化的潜能

目的探讨内皮祖细胞(EPCs)在不同诱导因子作用下的体外分化潜能。方法人脐血单个核细胞分别予50ng/ml血管内皮生长因子(VEGF组)或50ng/ml血小板源生长因子(PDGF组)诱导分化。光镜形态观察,免疫荧光鉴定。流式细胞分析CD133+EPCs分化特征。结果新鲜脐血分离单个核细胞培养1周后贴壁细胞的EPCs特异的DiI标记乙酰化低密度脂蛋白鉴定为80%阳性。在VEGF或PDGF诱导下1周,单个核细胞大量贴壁生长多呈圆形,少量梭形生长。2周时,被诱导细胞有近50%贴壁呈梭形生长,VEGF组和PDGF组无明显差异。至4周时两组出现明显的分化差异,其中VEGF组呈"铺路石"样细胞融合,血管性假血友病因子免疫荧光呈阳性;而PDGF组呈梭形或长多角形融合,予α-平滑肌肌动蛋白标记部分呈阳性。单个核细胞磁珠分选后得到CD133+较CD133-更多分化为内皮样细胞(P<0.05);而在PDGF的诱导下CD133-较CD133+更多分化为平滑肌样细胞(P<0.05)。结论单个核细胞在体外不同的诱导因子诱导下可以双向分化,CD133+EPCs具有更强的内皮细胞分化潜能。     

脐血外周血内皮祖细胞成内皮细胞的体外比较研究

目的:探讨人脐血、外周血内皮祖细胞(EPCs)体外分离、纯化、诱导扩增和分化为内皮细胞的可行性,并对两者分化成内皮细胞的能力进行比较。方法:新鲜脐血和健康成年人的外周血,Ficoll密度梯度离心法得单个核细胞,磁珠分选方法(MACS)分离脐血CD34 单个核细胞(MNCCD34 )。脐血单个核细胞(CBMC)、外周血单个核细胞(PBMC)和MNCCD34 在M-199培养基体外培养,血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)诱导EPCs增殖和分化。采用细胞形态学观察比较细胞克隆形成率的区别,流式细胞仪检测不同来源的EPCs分化后CD31阳性细胞率。结果:MNCCD34 培养3 d后,细胞克隆形成率为(24.25±6.5)/(3×107)细胞;CBMC贴壁细胞培养3 d后,为(103.00±10.10)/(3×107)细胞;PBMC贴壁细胞培养3 d后,为(74.25±5.44)/(3×107)细胞。CBMC分化形成的贴壁细胞和细胞簇数目多于PBMC(P<0.05),但单个核细胞分化形成的克隆率均明显高于MNCCD34 细胞(均P<0.05)。细胞培养10 d后CD31阳性率为:CBMC为(76.24±16.54)%,PBMC为(82.2±9.0)%,MNCCD34 为(70.03±10.27)%,MNCCD34-仅为(36.5±11.78)%。结论:相似数目的细胞,CD34 细胞单独培养形成的贴壁细胞和细胞簇数目明显少于CBMC和PB-MC(均P<0.05)。培养10 d后CD31阳性细胞率则差异无统计学意义(P>0.05),提示不同来源的EPCs分化率无显著区别,主要来自CD34 细胞群。     

内皮祖细胞及造血祖细胞在心血管疾病中的意义及研究进展

1 关于内皮祖细胞在心血管疾病中的研究 1.1 内皮祖细胞定义、表型特征及来源 内皮祖细胞(endothelial progenitor cells,EPCs)是一类具有特异性归巢于损伤区域并能分化增殖为成熟内皮细胞的一群干细胞.研究表明,EPCs可能来源于骨髓,在脐带血和外周血中也有存在.EPCs的表面标志尚未明确,一般认为CD34+KDR+细胞代表EPCs,而CD34(-)CD133(+)KDR(+)细胞代表更早期循环EPCs,具有更强的参与血管新生能力.1997年,Asahara等[1]首次在中分离出CD34+及ilk-l-的单个核细胞,研究证明这些细胞在体外能够参与成年动物血管的形成,具有EPCs的功能.此后,Shi等[2]也在人的骨髓、脐带血、10 ～ 15 w胎肝及外周血中分离了较高纯度的CD34(+)细胞.随后的一些研究中研究者又从异基因骨髓移植手术后的受者外周血中分离出单个核细胞,处理后观察发现具有内皮细胞特征的梭形细胞.种种实验研究表明,EPCs来源于骨髓、脐带血及外周血液等.     

人骨髓间充质干细胞向血管内皮细胞的诱导分化

目的观察人骨髓间充质干细胞（HBMCs）的体外培养及向血管内皮细胞的诱导分化结果。方法利用密度梯度离心法将HBMCs从人骨髓中分离出来,体外扩增。将培养至第3代的HBMCs加入含血管内皮生长因子（VEGF）、成纤维细胞生长因子（bFGF）的培养基定向诱导,使其向血管内皮细胞分化。用免疫细胞化学和流式细胞学方法检测诱导后的细胞表型。结果原代HBMCs培养3周后细胞呈梭形,诱导后第7天的细胞呈椭圆形或不规则形,第14天细胞大致呈＂铺路石＂样改变,细胞化学方法检测发现其CD31、CD34、vWF因子呈阳性,流式细胞仪测定CD31、vWF因子阳性细胞分别占87.5%、82.6%,CD31、vWF因子均阳性的细胞占71.2%。结论 HBMCs在VEGF、bFGF的诱导下向血管内皮细胞方向分化。     

骨髓CD34^＋干细胞向内皮细胞转化的诱导方法

目的探讨骨髓CD34^＋细胞向血管内皮细胞转分化的诱导方法。方法采集犬骨髓,经免疫磁珠分离出内皮祖细胞,内皮细胞生长因子（VEGF）诱导分化为内皮细胞并扩增,倒置相差显微镜、免疫细胞化学和摄取DilAc—LDL试验鉴定。将所得细胞种植于人工血管,扫描电镜观察细胞形态,并与MNCs作对比。结果经流式细胞仪测定,分离后的细胞中CD34^＋细胞占78．46％±6．37％;CD34^＋细胞培养2周后细胞基本铺满培养瓶底面,细胞呈“鹅卵石”状排列,CD34^＋和Ⅷ因子免疫细胞化学染色均为阳性。扫描电镜下观察可见内皮细胞平铺于人工血管表面,有伪足伸出并长入血管内表面微孔内。结论通过免疫磁珠方法可分离得到高纯度的骨髓CD34^＋细胞,经体外培养VEGF诱导后可定向分化为内皮细胞。     

Irradiation induces homing of donor endothelial progenitor cells in allogeneic hematopoietic stem cell transplantation

Functional abnormalities of the endothelial system may be caused by allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study is to explore the possibility that endothelial progenitor cells (EPCs) can be used in endothelial repair post-HSCT. EPCs were isolated from mouse bone marrow by density centrifugation and differential adherence. Numbers of endothelial cells (ECs) (CD31⁺CD133⁻CD45⁻), EPCs (CD31⁺CD133⁺–CD45^low/−) and carboxyfluorescein succinimidyl ester (CFSE)-positive cells in peripheral blood, spleen and bone marrow were determined at various time points by flow cytometry. The distribution of labeled EPCs was observed by fluorescence microscopy; morphological alterations of tissues were assessed by light microscopy and transmission electron microscopy. In the irradiated group, the numbers of circulating ECs and EPCs were elevated after pre-conditioning, reaching peaks at days 3 and 5; the counts remained high for about 5 days. In addition, CFSE-labeled cells were visualized in tissue and bone marrow. In conclusion, these results suggest the following: (a) the EPCs derived from mouse bone marrow mononuclear cells express phenotypes characteristic of normal EPCs, (b) irradiation during preconditioning damaged the endothelium, which initiated mobilization of EPCs, and (c) injury to the endothelium also caused extrinsic EPCs home to the damaged tissue.     

成人骨髓源内皮祖细胞的体外分离培养及鉴定

目的探讨成人骨髓源内皮祖细胞（EPCs）体外分离培养的可行性。方法抽取成年男性骨髓,体外全骨髓培养,传代后采用免疫微珠分选方法收集CD南细胞,EGM—MV2条件培养基诱导培养EPCs,观察细胞形态、生长情况;采用流式细胞技术检测分选细胞纯度,免疫荧光法检测EPCs特殊分子标志物CD34,CD133和VE-cadherin表达,透射电子显微镜观察分选细胞超微结构,UEA-1和Dil-ac-LDL双染色法检测分选细胞的吞噬功能。结果分选后细胞培养第3天出现集落样生长,集落边缘细胞形态伸展呈梭形或多边形,传代后呈现串珠样排列;培养至5～6d,细胞连接成大片条索状结构;CD34^＋、CD133^＋细胞百分率分别为24．13％、93．29％,其表面特异性表达CD133、CD34、VE-cadherin,具有EPCs形态特征,能吞噬Dil-ac-LDL并结合UEA-1。结论成人骨髓来源的EPCs经体外培养后形态、增殖率、生存能力、表面标志表达、功能等均较为稳定,可作为心血管组织工程种子细胞或用于干细胞治疗。     

Endothelial progenitor cells: isolation and characterization

Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs). Of the three cell markers (CD133, CD34, and the vascular endothelial growth factor receptor 2) that characterize the early functional EPCs, located predominantly in the bone marrow, EPCs obviously lose CD133/CD34 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor when migrating to the circulation. Various isolation procedures of EPCs from different sources by using adherence culture or magnetic microbeads have been described, but published findings with regard to the number of EPCs in the peripheral circulation of healthy adults are scanty and no data regarding the lifetime of EPCs in vivo exist. Clinical studies employing EPCs for neovascularization of ischemic organs have just been started; however, the mechanisms stimulating or inhibiting the differentiation of bone marrow-derived EPCs in vivo and the signals causing their adhesion, migration, and homing to sites of injured tissue are largely unknown at present.     

Endothelial progenitor cells in infantile hemangioma

Infantile hemangioma is an endothelial tumor that grows rapidly after birth but slowly regresses during early childhood. Initial proliferation of hemangioma is characterized by clonal expansion of endothelial cells (ECs) and neovascularization. Here, we demonstrated mRNA encoding CD133-2, an important marker for endothelial progenitor cells (EPCs), predominantly in proliferating but not involuting or involuted hemangioma. Progenitor cells coexpressing CD133 and CD34 were detected by flow cytometry in 11 of 12 proliferating hemangioma specimens from children 3 to 24 months of age. Furthermore, in 4 proliferating hemangiomas, we showed that 0.14% to 1.6% of CD45(-) nucleated cells were EPCs that coexpressed CD133 and the EC marker KDR. This finding is consistent with the presence of KDR(+) immature ECs in proliferating hemangioma. Our results suggest that EPCs contribute to the early growth of hemangioma. To our knowledge, this is the first study to show direct evidence of EPCs in a human vascular tumor.     

Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133(+) bone marrow cells, a subset of CD34(+) haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 +/- 5%), the CD133(+) bone marrow cells were grown on fibronectin-coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF-1). The CD133(+) fraction contained 95 +/- 4% CD34(+) cells, 3 +/- 2% cells expressing VEGF receptor (VEGFR-2/KDR), but did not express von Willebrand factor (VWF), VE-cadherin, P1H12 or TE-7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 +/- 5 times. The cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45(-) and CD14(-), and expressed several endothelial markers (UEA-1, VWF, P1H12, CD105, E-selectin, VCAM-1 and VE-cadherin) and typical Weibel-Palade bodies. They had a high proliferative potential (up to a 2400-fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co-cultivated with CD34(+) cells, in parallel with a purified fibroblastic cell monolayer. CD34(+) cells (10 x 10(5)) gave rise to 17,951 +/- 2422 CFU-GM colonies when grown on endothelial cells, and to 12,928 +/- 4415 CFU-GM colonies on fibroblast monolayers. The ECs also supported erythroid blast-forming unit (BFU-E) colonies better. These results suggest that bone marrow CD133(+) progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo-angiogenesis.     

Endothelial progenitor cells: mobilization,differentiation, and homing

Postnatal bone marrow contains a subtype of progenitor cells that have the capacity to migrate to the peripheral circulation and to differentiate into mature endothelial cells. Therefore, these cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. In general, EPCs are characterized by the expression of 3 markers, CD133, CD34, and the vascular endothelial growth factor receptor-2. During differentiation, EPCs obviously lose CD133 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor. EPCs seem to participate in endothelial repair and neovascularization of ischemic organs. Clinical studies using EPCs for neovascularization have just been started; however, the mechanisms stimulating or inhibiting the differentiation of EPC in vivo and the signals causing their migration and homing to sites of injured endothelium or extravascular tissue are largely unknown at present. Thus, future studies will help to explore areas of potential basic research and clinical application of EPCs.     

内皮祖细胞移植治疗大鼠急性心肌梗死的实验研究

目的探讨大鼠内皮祖细胞(EPCs)移植于梗死心肌的增殖分化情况及对心功能的影响。方法分离培养大鼠EPCs,免疫荧光法检测其CD34+、CD133+和Flk-1+的表达。将SD大鼠冠状动脉左前降支结扎制造急性心肌梗死模型后,在梗死心肌处植入DAPI标记的EPCs(实验组)或M 199培养液(对照组)。移植后1周及4周,心脏超声检查心功能,并对梗死区心肌组织进行移植细胞形态学检查及毛细血管密度测定,逆转录聚合酶链反应及酶联免疫吸附测定梗死周边区血管内皮生长因子(VEGF)的表达。结果培养获得EPCs,其表型为CD34+、CD133+和Flk-1+。植入梗死区的EPCs可以分化为血管内皮细胞,实验组梗死心肌处血管密度较对照组明显增高(P<0.01),并且VEGF基因及蛋白表达在移植后1周均较对照组明显增高(P<0.01)。移植后4周,实验组大鼠左心室射血分数及左心室短轴缩短率较对照组明显提高(P<0.01);而移植后1周,两组大鼠超声检测心功能各指标变化不明显。结论同种异体EPCs移植到梗死心肌大鼠缺血心肌能分化为毛细血管内皮细胞,促进梗死后心肌血管新生,改善心功能。