兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞(endothelial progenitor cell,EPCs)的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被(FN)的组织工程血管支架上,加入血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的"铺路石样"外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示:加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示:内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和纤维连接蛋白(FN)的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

巴曲酶可影响人外周血内皮祖细胞定向分化及增殖能力

背景：近年来内皮祖细胞促进机体血管新生方面的重要作用已形成共识,但内皮祖细胞的定向分化和扩增的问题是影响其疗效和广泛开展的主要原因。 目的：观察人外周血内皮祖细胞体外分离、纯化、扩增和诱导分化为内皮细胞的可行性。 方法：用密度梯度离心法从外周血获取单个核细胞,接种于包被有人纤维连接蛋白的细胞培养板中,培养6 d后,收集贴壁细胞。以血管内皮细胞生长因子和碱性成纤维细胞生长因子对贴壁细胞进行诱导培养。另外,分别以0.05,0.1,0.2 BU/mL巴曲酶培养贴壁细胞,观察内皮祖细胞的增殖能力。 结果与结论：经血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导后的细胞形态上表现内皮细胞特征,经流式细胞仪测定表达血管内皮细胞特有表面标志CD31和vWF,说明诱导后的细胞为内皮细胞。巴曲酶在体外培养条件下可增加血内皮祖细胞的数量,以0.1 BU/mL浓度作用显著,且作用时间与血内皮祖细胞增殖能力的改善呈正相关。     

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。     

人胚胎主动脉血管内皮祖细胞的分离、培养及鉴定

目的研究人胚胎血管内皮祖细胞(EPCs)分离、扩增及鉴定的方法,并评价其分化成血管内皮细胞的能力,为人胚胎血管来源EPCs作为干细胞技术治疗疾病的细胞材料提供依据。方法从14周龄流产人胚胎主动脉中应用胶原酶消化法分离获得EPCs,用含有碱性成纤维细胞生长因子、表皮生长因子和白血病抑制因子的低血清培养基体外扩增培养EPCs。分离培养的EPCs鉴定采用细胞免疫荧光染色、RT-PCR及流式细胞术,检测EPCs细胞的特异标志CD133、CD34和血管内皮细胞生长因子受体2(VEGFR2)。培养的EPCs应用VEGF进行诱导分化,并评价其分化成为血管内皮细胞的能力。结果分离的人胚胎主动脉EPCs细胞表达EPCs的标志分子CD133、CD34和VEGFR2。EPCs在体外特定低血清培养条件下表现很强的增殖能力。培养的EPCs细胞经过VEGF诱导后,细胞表达CD133明显降低,表达vWF、CD31和ELAM-1增强,并且体外成管能力和摄取Ac-LDL能力增强,表明细胞分化成为血管内皮细胞。结论人胚胎早期主动脉的EPCs具有很好的体外自我更新能力和分化成为血管内皮细胞的潜能,可作为EPCs治疗疾病的细胞材料。     

人外周血内皮祖细胞的培养过程及分化

目的：研究人外剧血中内皮祖细胞（endothelial progenitor cell,EPC）的培养及分离,并探索EPC生物学特性及诱导分化条件：方法：健康成人肘静脉血,用密度梯度离心法得到单个核细胞,接种于纤维连接蛋白包被的6孔板进行培养（含有人血管内皮细胞生长因子）,观察细胞集落、梭形贴壁细胞的形成过程。应用激光共聚焦显微镜和流式细咆仪进行EPC的鉴定。结果：在细胞培养的第4天,人外周血来源的单个核细胞开始形成从中间向外剧放射的细胞集落;住第7天时形成典型的长梭形,首尾相连成条索状;培养第2周时,长悛形细胞渐消失,出现鹅卵石样的圆形椭圆形细胞;在第3周时,鹅卵石样细胞增殖旺盛。可以应用激光共聚焦显微镜成功鉴定EPC,并且VEGF和人纤维连接蛋白可以促进EPC的生长。结论：人外周血中的内皮祖细胞来源于单个核细胞,早期内皮祖细胞出现于细胞培养第4-7天,晚期内皮祖绌胞出现于细胞培养2-3周时。EPC任特定条件下可分化为内皮细胞,为EPC的进一步研究及临床应用奠定了基础。     

人外周血单个核细胞来源内皮祖细胞的生物学性状

目的 探讨从成人外周血单个核细胞中分离、培养内皮祖细胞（EPCs）的方法以及EPCs的生物学特征。方法 密度梯度离心法获得单个核细胞（MNCs），种植于纤连蛋白包被的培养板中。每2h去除1次未黏附细胞共2次，然后隔日换液1次，7d后计数早期集落。每例血样均分为2等份，1份在获得早期集落后进行实验；另1份持续培养至晚期集落出现进行相同实验。流式细胞技术检测细胞表面抗原表达，ELISA法测定上清液中血管内皮生长因子（VEGF）浓度。胶原凝胶细胞种植实验测定体外血管生成功能。结果 早期集落中心为圆形细胞，周边是放射状排列的纺锤形细胞，再种植不能形成第二代集落且无体外血管形成功能，细胞表面主要表达CD14和CD45，培养上清液中可测到高浓度的VEGF。晚期集落在培养21至28d间出现，再种植可形成第二代内皮细胞集落，并能在胶原凝胶中形成管腔样结构。其构成细胞与早期集落相比CD45、CD14表达显著减少（P<0.001）而CD146表达明显增加（P<0.01）。结论 人外周血单个核细胞在内皮培养条件下可形成早期和晚期集落，构成早期集落的绝大多数细胞属于单核细胞系列，可分泌VEGF但不能分化成内皮细胞，晚期集落具有内皮祖细胞的形态和生物学特征。     

大鼠骨髓内皮祖细胞的分离、培养及鉴定

目的 研究大鼠骨髓来源的内皮祖细胞(EPC)的分离、鉴定、培养方法以及向内皮细胞分化的诱导条件.方法 密度梯度离心法分离SD大鼠股骨及胫骨单个核细胞,经差速贴壁后取2次贴壁细胞置于纤连蛋白铺被的培养板中.在血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)及表皮生长因子(EGF)诱导下培养两周,观察其形态学改变,以免疫细胞化学染色及Dil-acLDL、FITC-UEA-1双荧光染色法对其进行鉴定.结果 贴壁细胞呈现铺路石、团簇样生长,呈梭形、线样排列特殊形态.免疫细胞化学结果显示,贴壁细胞CD133、CD34、Flk-1、血管性假血友病因子(vWF)在不同时段呈阳性表达.诱导培养第2天,CD133阳性表达率为(79.4±4.5)%.自诱导培养的第6天开始,Flk-1与vWF呈高表达,阳性率为(74.2±3.5)%和(72.2±4.3)%.结论 用Percoll密度梯度离心法在VEGF、bFGF及EGF培养体系下可以获得较高纯度的EPC,该细胞具有内皮细胞的特性,经过体外诱导可以分化为内皮细胞.     

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。     

人骨髓间充质干细胞体外分离培养及向血管内皮样细胞分化的研究

目的:研究人骨髓间充质干细胞(hMSCs)的体外分离培养方法及在特定微环境下分化为血管内皮样细胞的潜能,可能为银屑病研究提供实验基础.方法:密度梯度离心法结合贴壁法分离人骨髓间充质干细胞(hMSCs),体外扩增培养并进行鉴定.加入血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)定向诱导hMSCs向血管内皮细胞分化,流式细胞仪进行细胞表型鉴定,Dil-ac-LDL摄取实验鉴定血管内皮细胞功能.结果:经流式细胞仪测定分离培养的hMSCs CD71、CD44阳性表达,CD54、CDl06、CD45弱阳性表达,CD34、CD31、VWF、KDR、HLA-DR阴性表达;经bFGF、VEGF诱导后的hMSCs可见类似内皮细胞样改变,经流式细胞术检测,与对照组相比内皮细胞特异表面标志CD34、CD31、VWF、KDR表达转为阳性(P＜0.01),而HLA-DR、CD54、CDl06、CD45表达明显上调(P＜0.01);诱导分化后的内皮样细胞具有摄取Dil-ac-LDL的能力.结论:密度梯度离心法结合贴壁法可获得较纯的MSCs;在bFGF、VEGF诱导作用下,hMSCs具有向内皮细胞分化潜能,产生功能性内皮细胞.     

用CD133免疫磁珠分离脐血内皮祖细胞的实验研究

目的：从脐血中分离、培养血管内皮祖细胞,研究内皮祖细胞的生长特性和诱导分化条件。 方法： 应用MACS磁球抗体标记法纯化脐血中的CD133+细胞，通过流式细胞仪、免疫细胞化学、免疫荧光等技术及形态学（光镜、电镜）观察研究内皮祖细胞；将细胞接种于添加（或未添加）VEGF、bFGF、干细胞因子（SCF）的含20％胎牛血清（FBS）的IMDM培养基中,观察内皮祖细胞的生长特性。 结果： 分离新鲜脐血所得CD133阳性细胞占单个核细胞的(1.41±1.14)%,经流式细胞仪鉴定CD133+细胞纯度为75%-85%；将分离细胞接种于纤维连接蛋白包被的24孔板内，培养1-2 h即有细胞贴壁，7-10 d可见贴壁细胞呈铺路石样排列；14 d后细胞出现小圆形、梭形等多样性变化，可见毛细血管管腔样结构，电镜观察可见胞浆内典型的Weibel-Palade小体；在VEGF、bFGF、SCF存在条件下,检测贴壁细胞培养14 d后细胞表面抗原表达情况：与培养开始时相比，祖细胞标志CD133和CD34阳性率呈明显下降趋势，分别由(77.0±3.3)%和(93.1±4.7)%降至(1.6±2.2)%和(37.4±4.9)%，P<0.05，内皮细胞特异性标志Flk-1表达明显增加，由(22.3±3.3)%增至(94.3±4.1)%，P<0.05，同时vWF抗原呈强阳性表达，阳性率为(77.9±3.3)%。 结论： 根据细胞表面特异性分子标志（CD133+/CD34+/Flk-1+）可以从脐血中分离出EPCs，EPCs可在体外一定的诱导因子作用下，培养7-10 d分化为成熟内皮细胞。     

Accelerating vascularization in polycaprolactone scaffolds by endothelial progenitor cells

Vascularization is a major challenge in tissue engineering. The purpose of this study is to expedite the formation of blood vessels in porous polycaprolactone (PCL) scaffolds by the delivery of endothelial progenitor cells (EPCs). To establish a pro-angiogenic and pro-vasculogenic microenvironment, we employed EPCs seeded in PCL scaffold with surface-immobilized heparin and vascular endothelial growth factor (VEGF). EPCs seeded on scaffolds with VEGF exhibited phosphorylation of the receptor. After 7 days of subcutaneous implantation in immunodeficient mice, heparin-immobilized PCL scaffolds with VEGF induced significantly high density of blood vessel formation. The anastomosis of EPC-derived vessels with the host circulatory system was evident by the presence of murine erythrocytes in the lumen of human-CD31 positive vessels. A more uniform distribution of blood vessels was achieved within 2-mm thick scaffolds by seeding an optimal density of EPCs. The seeding of a higher density of EPC resulted in an increase in apoptosis and a concomitant decline in blood vessel formation at the scaffold's inner core. When co-seeded with other cells, the EPCs maintained the ability to accelerate vessel formation. The excessive expansion of EPCs in vitro was associated with a decline in their in vivo vasculogenic potential. EPCs accelerated the vascularization of heparin-immobilized PCL scaffolds in the presence of VEGF.     

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。     

Human endothelial progenitor cell-seeded hybrid graft: proliferative and antithrombogenic potentials in vitro and fabrication processing

In this article, we show that human endothelial progenitor cells (EPCs) in circulating peripheral blood may be a novel cell source for a cell-incorporated engineered vascular graft. Cultures of human peripheral blood mononuclear cells collected by the density gradient technique developed highly proliferative EPC colonies, which expanded with culture time. The production rates of antiplatelet substances such as endothelial-type nitric oxide synthase and 6-keto-prostaglandin-F(1)-alpha were approximately one-third and approximately one-half of those of mature endothelial cells (ECs), respectively. On the other hand, the tissue-type plasminogen activator production rate of EPCs was almost the same as that of ECs. EPCs were seeded and cultured on a small-diameter compliant graft (inner diameter, 1.5 mm) made of microporous segmented polyurethane film coated with a photo-reactive gelatin layer, and subsequently subjected to hydrodynamic shear stress by ex vivo circulation. EPCs fully covering the graft elongated and aligned themselves with the direction of the flow, resulting in the production of an integrated EPC-engineered graft. These results indicate that EPCs, which have high proliferative potential and high antithrombogenic potential, comparable to those of ECs, are a suitable cell source for cardiovascular tissue engineering.     

内皮祖细胞在体外培养成血管样结构的初步观察

目的：探索体外培养脐血、外周血内皮祖细胞（EPCs）的方法，观察其形成血管样结构的可能性及条件。 方法： 采用贴壁选择法培养人脐血及兔外周血内皮祖细胞，光镜下观察细胞形态，用荧光显微镜、流式细胞仪分析贴壁细胞CD34、VEGFR-2、AC133、血管内皮钙粘素（VE-cadherin）的表达，DiI-ac-LDL 吞噬试验及Ⅷ因子免疫组化证实细胞属性。 结果： 体外成功培养出人脐血及兔外周血内皮祖细胞，形成条索状、管状结构，兔外周血EPCs分化较成熟，形成典型铺路石形状及血管样结构。 结论： 脐血、兔外周血内皮祖细胞可在体外培养成功并表现成血管倾向，可能是血管组织工程的潜在资源。     

定向诱导骨髓间充质干细胞参与创面修复的研究

目的探讨诱导骨髓间充质干细胞(MSCs)分化为血管内皮样细胞参与创面修复的可能机制。方法选择正常成人MSCs,经密度梯度离心分离、纯化及体外培养扩增后,定向诱导分化为血管内皮样细胞作为种子细胞,以大于(1～2)×10个/cm密度种植于支架上,扫描电镜观察后,应用溴脱氧尿嘧啶(BrdU)标记技术标记细胞。于兔子背部制作全层皮肤缺损创面,即刻以纤维蛋白胶为载体,将已标记的血管内皮样细胞回植到供体动物创面上。术后2周切取创面组织,行BrdU和凝血因子Ⅷ(FⅧ)免疫组织化学染色。结果 BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围,且有个别血管内皮细胞也呈现BrdU阳性。部分MSCs细胞质中亦有FⅧ表达。结论创面愈合过程中,诱导的MSCs与肉芽组织中小血管的形成密切相关。诱导的MSCs可分化为血管内皮样细胞,并参与创面修复。     

In vitro angiogenesis properties of endothelial progenitor cells: a promising tool for vascularization of ex vivo engineered tissues

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at the improvement of neovascularization of engineered tissues are a key issue. A method to enhance graft vascularization is to establish a primitive vascular plexus within the graft before transplantation by the use of cellular-based concepts. To explore the utility of endothelial progenitor cells (EPCs) for the ex vivo vascularization of tissue engineered grafts, we analyzed the in vitro angiogenic properties of this cell type in two different angiogenesis models: the 3-dimensional spheroid sprouting assay and the 2-dimensional matrigel assay. In both assays, EPCs were able to form tubelike structures, resembling early capillaries. This process was significantly enhanced by the addition of angiogenic growth factors. Direct comparison between EPCs and mature endothelial cells, represented by human umbilical vein endothelial cells (HUVECs), revealed that both cell types displayed an almost identical angiogenic potential. Other functional in vitro parameters such as angiogenic growth factor induced cell proliferation and cell survival were investigated as well, revealing a significantly decreased level of apoptosis of EPCs in relation to HUVECs under serum-deprived conditions. The observed survival advantage of EPCs along with the observation that EPCs perform very well in the above mentioned in vitro angiogenesis assays, make them an ideal autologous cell source for vascularization of ex vivo generated tissues. The attractiveness of this cell type for tissue engineering applications is strengthened further by the fact that these cells can be easily isolated from the peripheral blood of patients, thereby eliminating donor site morbidity.     

The effect of stromal cell-derived factor-1α/heparin coating of biodegradable vascular grafts on the recruitment of both endothelial and smooth muscle progenitor cells for accelerated regeneration

Small-diameter synthetic vascular grafts have high failure rate and tissue-engineered blood vessels are limited by the scalability. Here we engineered bioactive materials for in situ vascular tissue engineering, which recruits two types of endogenous progenitor cells for the regeneration of blood vessels. Heparin was conjugated to microfibrous vascular grafts to suppress thrombogenic responses, and stromal cell-derived factor-1α (SDF-1α) was immobilized onto heparin to recruit endogenous progenitor cells. Heparin-bound SDF-1α was more stable than adsorbed SDF-1α under both static and flow conditions. Microfibrous grafts were implanted in rats by anastomosis to test the functional performance. Heparin coating improved the short-term patency, and immobilized SDF-1α further improved the long-term patency. SDF-1α effectively recruited endothelial progenitor cells (EPCs) to the luminal surface of the grafts, which differentiated into endothelial cells (ECs) and accelerated endothelialization. More interestingly, SDF-1α increased the recruitment of smooth muscle progenitor cells (SMPCs) to the grafts, and SMPCs differentiated into smooth muscle cells (SMCs) in?vivo and in?vitro. Consistently, SDF-1α-immobilized grafts had significantly higher elastic modulus. This work demonstrates the feasibility of simultaneously recruiting progenitor cells of ECs and SMCs for in situ blood vessel regeneration. This in situ tissue engineering approach will have broad applications in regenerative medicine.