淋巴管内皮祖细胞在淋巴管新生中的作用及其机制

淋巴管新生在胚胎发育、外伤修复、炎症转归、肿瘤转移和器官移植后排斥反应等方面起着重要作用.除淋巴管内皮细胞外,淋巴管内皮祖细胞参与淋巴管新生.淋巴管内皮祖细胞表达CD34、CD133和VEGFR-3,在趋化因子作用下由骨髓动员入外周血,继而迁入局部组织.经VEGF-C等生长因子诱导向淋巴管内皮细胞分化,参与淋巴管新生....     

黑色素瘤细胞诱导骨髓间充质干细胞向血管内皮细胞分化的初步研究

目的 体外观察和分析小鼠黑色素瘤细胞B16诱导小鼠骨髓间充质干细胞(BMSC)向血管内皮细胞分化的现象和过程,初步分析肿瘤细胞分泌血管生长因子(VEGF)-a在该诱导过程中的时序关系.方法 利用Transwell系统进行BMSC与B16细胞共培养,利用免疫荧光、蛋白印迹、透射电镜等技术观察BMSC经B16诱导后向内皮细胞分化的过程,利用ELISA检测培养液内VEGF-a的水平.结果 BMSC表型鉴定为CD44+/CD73+/CD90+/CD105+/CD166+/CD34-/CIM5-/CD133-,经B16诱导后出现内皮细胞标志物VEGFR-1、VEGFR-2和第八因子(FⅧ)的表达并随时间延长而增强,共培养48 h后VEGFR-1、VEGFR-2及FⅧ均可见明显表达,72 h时VEGFR-2表达量较48 h时增加了1倍,FⅧ表达量增加了约3倍.共培养体系培养基中可检测到由B16细胞分泌的VEGF-a量随时间增多.结论 B16细胞可能通过分泌VEGF-a来诱导BMSC具备血管内皮细胞表型,提示BMSC在肿瘤血管生成中起了重要的作用.     

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。     

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。     

犬骨髓内皮祖细胞生物学特性及诱导分化

目的:探讨犬骨髓内皮祖细胞(EPCs)随培养时间延长其生物学特性变化及向内皮细胞(ECs)分化能力。方法:免疫微珠分选方法纯化犬骨髓CD14 细胞,EGM-MV2条件培养基培养,于不同时间检测EPCs的生物学特性;将存活至8周的EPCs以分化培养基(M199 20%胎牛血清 VEGF)诱导培养,检测ECs表面标志的表达。结果:早期EPCs为分选后8代之前的EPC,体积略小,类圆形,60%融合时呈串珠样排列,表达CD133、CD14;晚期EPCs由表达VE-cadherin、KDR、CD14的早期EPCs分化而来,形态为多边形,分泌活动明显加强,表达CD133、VE-cadherin、CD14和KDR;诱导分化细胞呈鹅卵石样外观,表达Ⅷ因子、CD31。结论:犬骨髓EPCs随培养时间呈现不同生物学特性,早期EPCs不稳定,晚期EPCs显示出更稳定的生物学特性,能够分化为ECs,是血管组织工程学研究良好的种子细胞。     

人动脉血内皮祖细胞的分离与鉴定

<正>血管内皮祖细胞(endothelial progenitor cells,EPCs)与胚胎发育中血管母细胞可能存在延续关系,可以诱导分化为血管内皮细胞,而且在体内血管新生过程中也发挥重要作用~([1])。应用EPCs移植治疗心血管疾病正在成为目前研究的热点。以往研究中EPCs主要来源于骨髓、脐带血、外周血以及脂肪组织。外周血来源的EPCs,主要从静脉血中进行分离,动脉血中分离     

人脐血源性内皮祖细胞血管发生活性及其参与人恶性胶质瘤细胞异种移植瘤血管新生的作用

目的 观察人脐血源性内皮祖细胞(EPC)血管发生能力和在恶性胶质瘤血管新生过程中的作用.方法 应用密度梯度离心法分离新鲜人脐血的单个核细胞,接种于EGM-2培养液中培养获得EPC.取生长到第7～10天的细胞进行CD34和VEGFR-2免疫荧光双标染色.检测血管内皮生长因子(VEGF)刺激下EPC增殖活性、迁移能力和体外形成小管样结构的能力.采用人恶性胶质瘤细胞系U87在免疫缺陷小鼠进行皮下移植,于接种肿瘤后第7天经尾静脉注射EPC(每只5×103),并于接种肿瘤后第28天取材检测肿瘤微血管和EPC组织分布及定位,采用抗人CD31和抗鼠CD31免疫荧光双标记肿瘤微血管,计算人源性EPC来源的血管占肿瘤血管网的比例.结果 培养的细胞在第7～10天时可见条索样结构,生长并逐渐融合形成铺路石样排列的单层细胞,表达内皮细胞标记物CD34和VEGFR-2.在VEGF刺激下EPC具有较强的增殖活性、迁移能力和体外形成小管样结构的能力.外源性EPC能特异性归巢到异种移植瘤组织并形成新生血管,占肿瘤血管网的(18.68±1.32)%.结论 EPC在体内外具有形成血管能力,并参与异种移植瘤血管新生,提示其在恶性肿瘤血管新生过程中具有重要作用,并可能参与肿瘤微血管构筑表型异质性.     

从PBMCs及CD133免疫磁珠分选获得内皮祖细胞的体外研究

目的：比较从外周血单个核细胞(PBMCs)和CD133免疫磁珠分选体外培养人外周血内皮祖细胞(EPCs)的表型、分化、扩增、功能特点,为EPCs细胞治疗提供临床参考。方法： 健康成年人外周血,经Ficoll密度梯度离心法得PBMCs,经直接接种法或免疫磁珠分选CD133阳性细胞后置于M199培养基中培养,于第7、14 d比较2组细胞表型变化及细胞因子分泌、扩增、体外血管形成及趋化能力。结果：相同血量标本PBMCs组早期集落数高于CD133+组(P<0.01),随着培养时间的推移,流式细胞检测2组细胞均显示造血干细胞标志的表达下调和内皮细胞标志表达上升,但CD133+组内皮标志CD144表达率低于PBMCs组(P<0.01),ELISA法检测到在PBMCs组早期EPCs分泌VEGF的水平高于CD133+组(P<0.01),MTT法显示PBMCs组有较强的增殖能力,基质胶及Transwell实验表明PBMCs组细胞参与血管形成的能力较强。结论： CD133+来源的EPCs分化、分泌、增殖及血管形成能力相对较低,推断PBMCs中CD133-细胞可能在形成功能性的EPCs中发挥更重要的作用。     

CD133+卵巢癌干细胞样细胞在血管内皮细胞中的分化

背景：大量研究证实,新生血管形成在肿瘤的生长、浸润以及转移过程中发挥重要作用。 目的：探讨CD133+卵巢癌干细胞样细胞向血管内皮细胞分化的特点。 方法：通过无血清培养方法从卵巢癌A2780细胞株中成功诱导出CD133⁺卵巢癌干细胞样细胞,在体外接种于铺或不铺Matrigel基质胶的96孔板内,观察不同时间点CD133⁺卵巢癌干细胞样细胞和人脐静脉内皮细胞形成管腔样结构能力。通过裸鼠皮下移植实验,免疫荧光法观察CD133⁺卵巢癌干细胞样细胞在卵巢癌血管新生中的作用。 结果与结论：CD133⁺卵巢癌干细胞样细胞和人脐静脉内皮细胞(阳性对照)在未铺 Matrigel基质胶上并不能形成相应的管腔结构,且不表达内皮细胞标志物CD31,在Matrigel基质胶上能够形成相对稳定的管腔结构,CD31表达明显。CD133⁺卵巢癌干细胞样细胞接种裸鼠皮下成瘤后,可观察到肿瘤组织中有人源性CD31的表达。结果表明CD133⁺卵巢癌干细胞样细胞能够分化为血管内皮细胞,参与肿瘤血管重建。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

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。     

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。     

骨髓源性血管内皮前体细胞与出生后生理性和病理性血管形成

Endothelial progenitor cells （EPCs） exist in bone marrow, umbilical cord blood and peripheral blood of adult mammals, including humans. Furthermore, the discovery of EPCs has led to the notion of adult vasculogenesis, in which bone marrow (BM)-derived EPCs home to and incorporate into sites of new blood vessel formation, where they differentiate into endothelial cells, which is consistent with postnatal vasculogenesis. It has become apparent that circulating BM-derived EPCs are involved in promoting physiologic andpathologic neovascularization, such as wound healing and tumor growth. They are of great clinical importance in pro-or anti-angiogenic therapies.     

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。     

Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization

A subset of human peripheral blood mononuclear cells (PB-MNCs) differentiate into endothelial progenitor cells (EPCs) that participate in postnatal neovascularization. Although tissue ischemia can mobilize EPCs from bone marrow, the effects of hypoxia on differentiation and angiogenic function of EPCs are little known. We examined whether hypoxic conditioning would modulate differentiation and function of human PB-MNC-derived EPCs. A subset of PB-MNCs gave rise to EPC-like attaching (AT) cells under either normoxic or hypoxic conditions. However, hypoxia much enhanced the differentiation of AT cells from PB-MNCs compared with normoxia. AT cells released vascular endothelial growth factor (VEGF) protein and expressed CD31 and kinase insert domain receptor/VEGFR-2, endothelial lineage markers, on their surface, which were also enhanced by hypoxia. Both a neutralizing anti-VEGF mAb and a KDR-specific receptor tyrosine kinase inhibitor, SU1498, suppressed PB-MNC differentiation into EPC-like AT cells in a dose-dependent manner. Migration of AT cells in response to VEGF as examined by a modified Boyden chamber apparatus was also enhanced by hypoxia. Finally, in vivo neovascularization efficacy was significantly enhanced by in vitro hypoxic conditioning of AT cells when cells were transplanted into the ischemic hindlimb of immunodeficient nude rats. In conclusion, hypoxia directly stimulated differentiation of EPC-like AT cells from human PB-MNC culture. Moreover, hypoxic preconditioning of AT cells before in vivo transplantation is a useful means to enhance therapeutic vasculogenesis.     

内皮祖细胞在血管组织工程的应用

内皮祖细胞是一群具有游走特性,能进一步增殖分化成为成熟内皮细胞的幼稚内皮细胞.内皮祖细胞参与了出生后缺血组织的血管发生和血管损伤后的修复.内皮祖细胞的发现为血管组织工程种子细胞增添了一个新来源.本文重点介绍成体内皮祖细胞的来源、鉴定、生物学特性、功能以及在血管组织工程中的应用.     

Role of bone morphogenetic protein 2 in the crosstalk between endothelial progenitor cells and mesenchymal stem cells

In recent studies, we and others have demonstrated that bone morphogenetic protein-2 (BMP-2) promotes vascularization, inhibits hypoxic cell death of cancer cells and may be involved in tumor angiogenesis. The activation of circulating endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) represents a crucial factor in the process of postnatal neovascularization. BMP-2 protein expression has been detected in several tumor tissues and BMP receptors are expressed in EPCs and MSCs. We therefore analysed the influence of recombinant human (rh) BMP-2 on the function of human EPCs and human bone marrow derived MSCs. Treatment of EPCs isolated from peripheral blood with rhBMP-2 did not induce any significant changes in EPC viability but induced a dose-dependent activation of chemotaxis. Incubation of human MSCs isolated from bone marrow aspirates with rhBMP-2 revealed no significant effect on MSC proliferation. Incubation of EPCs with supernatants of MSCs significantly increased the cell viability compared to controls cultivated with endothelial cell medium. Protein and mRNA expression of the vascular endothelial growth factor (VEGF) family member, placental growth factor (PlGF), which is known to be involved in the expansion and recruitment of EPCs, was induced in MSCs after treatment with rhBMP-2. We conclude that tumor- associated BMP-2 secretion might promote tumor angiogenesis by chemotactic effects on EPCs circulating in the peripheral blood and by increased secretion of paracrine angiogenic growth factors including PlGF in MSCs of the tumor stroma.     

Bone marrow-derived endothelial progenitor cells are a major determinant of nascent tumor neovascularization

Tumors build vessels by cooption of pre-existing vasculature and de novo recruitment of bone marrow (BM)-derived endothelial progenitor cells (EPCs). However, the contribution and the functional role of EPCs in tumor neoangiogenesis are controversial. Therefore, by using genetically marked BM progenitor cells, we demonstrate the precise spatial and temporal contribution of EPCs to the neovascularization of three transplanted and one spontaneous breast tumor in vivo using high-resolution microscopy and flow cytometry. We show that early tumors recruit BM-derived EPCs that differentiate into mature BM-derived endothelial cells (ECs) and luminally incorporate into a subset of sprouting tumor neovessels. Notably, in later tumors, these BM-derived vessels are diluted with non-BM-derived vessels from the periphery, which accounts for purported differences in previously published reports. Furthermore, we show that specific ablation of BM-derived EPCs with alpha-particle-emitting anti-VE-cadherin antibody markedly impaired tumor growth associated with reduced vascularization. Our results demonstrate that BM-derived EPCs are critical components of the earliest phases of tumor neoangiogenesis.     

Mechanisms underlying acceleration of blood flow recovery in ischemic limbs by macrophage colony-stimulating factor

Recently we reported that macrophage colony-stimulating factor (M-CSF) can mobilize endothelial progenitor cells (EPCs) from the bone marrow into the peripheral blood, resulting in an increase in the number of blood vessels and augmentation of blood flow in the ischemia-induced legs. M-CSF accelerates neovascularization of ischemic lesions resulting from the mobilization of EPCs. In the present paper, we analyze the mechanisms underling the mobilization of EPCs by M-CSF. M-CSF augments the production of vascular endothelial growth factor (VEGF) from the bone marrow cells, especially from myeloid lineage cells. In vivo administration of anti-VEGF antibody abrogates both the acceleration of the recovery of blood flow in the ischemia-induced limbs by M-CSF and the augmentation of the mobilization of EPCs induced by M-CSF. These results suggest that the M-CSF contributes to rapid recovery of blood flow in ischemic lesions by mobilization of EPCs from the bone marrow through augmentation of VEGF production in the bone marrow and that the VEGF is mainly produced by myeloid lineage cells.