体外诱导骨髓CD34+细胞生成血管内皮细胞的方法

目的：体外分离狗骨髓CD34^ 细胞，建立诱导分化血管内皮细胞的方法，并进行生物学鉴定。方法：利用免疫磁珠法分离狗骨髓CD34^ 细胞，在体外经血管内皮细胞生长因子（VEGF）、内皮细胞生长因子（EGF）、碱性成纤维细胞生长因子（hFGF)诱导后，观察细胞生长状况，以光镜、电镜进行形态学鉴定，应用免疫组化方法检测冯维靳布兰德因子（Von Willebrand factor,vWF)表达。结果：光镜下细胞单层融合生长，呈铺路石样形态，单个核位子中央，细胞群体倍增时间为35h。电镜下可见到Weibel-Palade小体，在细胞胞浆vWF染色阳性。结论：采用免疫磁珠法可从骨髓获得高纯度的CD34^ 细胞，在体外诱导分化成血管内皮细胞。     

绿色荧光蛋白转基因小鼠骨髓间充质干细胞向内皮细胞定向分化的能力

目的:研究绿色荧光蛋白转基因小鼠骨髓间充质干细胞(mesenchymastemcells,MSCs)体外扩增及其向内皮细胞定向分化能力,为血管组织工程在体研究提供实验依据。方法:实验于2003-10/2004-10在第三军医大学基础部解剖学教研室完成。无菌条件下采集绿色荧光蛋白转基因小鼠骨髓,用直接贴壁法获得小鼠MSCs,然后以血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)进行诱导分化,最后对扩增的细胞特征进行八因子(Willbrandfactor,vWF)免疫荧光鉴定观察。结果:MSCs每扩增一代,细胞数量增加4～8倍,7.0×103个原代MSCs体外扩增3代即获得1.4×108个细胞。在70%～80%融合时呈现“铺路石”样形态,说明绿色荧光蛋白转基因小鼠MSCs在体外的培养扩增条件与一般小鼠相同。添加VEGF诱导2周后,以免疫荧光法检测细胞的vWF的表达发现,诱导组的vWF呈阳性表达,对照组vWF表达呈阴性。结论:绿色荧光蛋白转基因小鼠MSCs在体外的培养扩增能力强,在VEGF作用下可以向内皮细胞转化。     

绿色荧光蛋白转基因小鼠骨髓间充质干细胞向内皮细胞定向分化的能力

目的：研究绿色荧光蛋白转基因小鼠骨髓间充质干细胞(mesenchvmal stem celis，MSCs)体外扩增及其向内皮细胞定向分化能力，为血管组织工程在体研究提供实验依据。方法：实验于2003—10／2004—10在第三军医大学基础部解剖学教研室完成。无菌条件下采集绿色荧光蛋白转基因小鼠骨髓，用直接贴壁法获得小鼠MSCs，然后以血管内皮生长因子(vascular endothelial growth faclor，vEGF)进行诱导分化，最后对扩增的细胞特征进行八因子(Willbrand factor，vWF)免疫荧光鉴定观察。结果：MSCs每扩增一代，细胞数量增加4～8倍，7．0&;#215;10^3个原代MSCs体外扩增3代即获得1．4&;#215;10^8个细胞。在70％～80％融合时呈现“铺路石”样形态，说明绿色荧光蛋白转基因小鼠MSCs在体外的培养扩增条件与一般小鼠相同。添加VEGF诱导2周后，以免疫荧光法检测细胞的vWF的表达发现，诱导组的vWF呈阳性表达，对照组vWF表达呈阴性。结论：绿色荧光蛋白转基因小鼠MSCs在体外的培养扩增能力强，在vEGF作用下可以向内皮细胞转化。     

间充质干细胞分化的内皮细胞有免疫调控作用

为了探讨间充质干细胞（mesenchymal stem cell，MSC）诱导而来的内皮细胞的免疫调控能力，将人骨髓MSC在体外经VEGF诱导分化为内皮细胞，并用倒置相差显微镜和流式细胞术分别观察和检测细胞的形态和免疫表型，用淋巴细胞转化实验观察其免疫调控能力。结果表明：MSC在向内皮细胞分化的体系中生长迅速，诱导后的细胞经von Willebrand factor（vWF）免疫荧光染色呈阳性，能在Matrigel上形成毛细血管样网状结构。细胞表面标记无明显改变，即CD73、CD105、HLA—ABC仍阳性，CD34、CD80、CD86、HLA—DR、CD31仍为阴性。由MSC诱导分化而来的内皮细胞能抑制T淋巴细胞的增殖，其抑制作用随内皮细胞加入量的增多而增强。结论：由骨髓MSC来源的内皮细胞对T淋巴细胞具有调控作用。     

人脂肪组织来源CD34＋／CD31-细胞向内皮细胞诱导分化的实验研究

目的 探讨将人脂肪组织来源CD34 /CD31-细胞诱导为内皮细胞的可行性.方法 脂肪组织来自5例接受脂肪抽吸术的患者,酶消化法获取原代细胞,细胞扩增培养至第二代,应用免疫磁珠仪分选CD34 /CD31-细胞.将此群细胞用VEGF及bFGF进行诱导.流式细胞仪检测分选前后细胞纯度;免疫细胞荧光检测细胞vWF和CD31的表达;荧光显微镜观察细胞摄取低密度脂蛋白的功能;三维培养观察细胞形成血管样结构的能力.结果分选前后CD34 /CD31-细胞比例分别为(12.57±1.83)%和(96.05±6.73)%;细胞诱导10d后呈现内皮细胞典型的"铺路石"样形态,vWF、CD31表达阳性,细胞具有摄取低密度脂蛋白的功能,三维培养条件下形成"树枝样"分叉结构.结论人脂肪组织来源的CD34 /CD31-细胞能够诱导分化为成熟内皮细胞.     

人胚胎干细胞接种在鼠尾胶原包被培养板上定向分化为血管内皮细胞

背景：胚胎干细胞在体外可诱导分化为血管内皮细胞,进而形成血管,因而成为血管组织工程理想的种子细胞来源之一。目的：探讨建立定向诱导人胚胎干细胞向血管内皮细胞分化的方法。方法：在小鼠胚胎成纤维细胞饲养层上培养人胚胎干细胞H1,将H1细胞团块转移到鼠尾胶原包被的培养板,贴壁24-48h后,培养基换为含不同辅助因子和内皮细胞生长添加剂的EGM-2内皮细胞培养基。结果与结论：①在EGM-2内皮细胞培养基诱导下,细胞逐步表达内皮细胞特异性标记基因VEGFR-2、PECAM1、vWF、CD34、VE-cadherin、GATA-2。②分化细胞表达内皮细胞特异性标记VE-cadherin、CD31。③分化细胞具有吞噬低密度脂蛋白功能。说明将人胚胎干细胞接种在鼠尾胶原包被培养板上,通过EGM-2内皮培养体系诱导,可直接分化为功能性血管内皮细胞。为研究胞外基质对诱导胚胎干细胞血管内皮细胞分化的作用以及相关信号分子机制打下了基础。     

人骨髓CD34＋干细胞分离培养及血管内皮生长因子165诱导分化的作用

背景:有研究证实,骨髓中CD34+干细胞在一定条件下具有向内皮细胞分化的潜能.目的:体外分离、纯化、培养和扩增人CD34+造血干细胞,并检测其免疫学表型,观察血管内皮生长因子对其体外诱导分化后细胞免疫表型的变化.设计、时间及地点:细胞学观察实验,于2005-10/2007-10在南方医科大学神经生物学教研室完成.材料:骨髓来源于健康供者.方法:利用Percoll梯度分离、贴壁筛选法及单克隆培养法分离培养、扩增人骨髓CD34+干细胞.采用脂质体介导转染法,选生长良好的传代细胞,以血管内皮生长因子165基因转染人骨髓CD34+干细胞.主要观察指标:采用免疫荧光和流式细胞术检测人骨髓CD34+干细胞免疫学表型.经血管内皮生长因子165基因转染后,人骨髓CD34+干细胞的表型变化以及血管内皮生长因子的分泌情况.结果:体外分离培养出高度同源性的人骨髓CD34+干细胞,细胞形态呈成纤维细胞样.CD44、CD29和c-kit阳性,CD31和CD54阴性;经血管内皮生长因子165诱导后,人骨髓CD34+干细胞表面标志CD44表达降低,CD31升高,呈现典型的内皮细胞表型,并且获得大量分泌血管内皮生长因子的能力.结论:采用Percoll分离液梯度离心继以贴壁筛选法及单克隆培养法联合筛选分离,可培养扩增}H高度同源的CD34+干细胞,经血管内皮生长因子基因转染后,CD34+干细胞呈典型的内皮细胞表型,验证了其具有向内皮细胞分化的潜能.     

动脉再生机制研究：骨髓间充质干细胞诱导分化内皮样细胞的体外实验

目的：探讨骨髓间充质干细胞体外扩增及其向内皮样细胞定向分化条件。方法：无菌条件下采集兔骨髓，肝素抗凝，经淋巴细胞分层液(相对密度为1.077)密度梯度离心分离骨髓单个核细胞，通过贴壁培养法获得骨髓间充质干细胞(bone marrow-mesenchymal stem cell，MSC)，然后在内皮细胞生长环境中进行诱导分化，观察细胞的体外生长特点及其生化特性。结果：MSC每扩增一代，细胞数量增加5～8倍，7.65&;#215;10^3个原代MSCs体外扩增3代即获得1.26&;#215;108个细胞。特定条件下贴壁细胞延展呈梭形，形成细胞簇、索状结构及“铺路石”样结构，扩增细胞免疫组化证实表达CD31和VIII因子相关抗原(factor VIII related antigen von Wille-brand factor，vWF)。具有吞噬乙酰化低密度脂蛋白的功能。结论：．MSC扩增能力强，在体外能诱导其向内皮细胞方向分化。     

动脉再生机制研究:骨髓间充质干细胞诱导分化内皮样细胞的体外实验

目的:探讨骨髓间充质干细胞体外扩增及其向内皮样细胞定向分化条件。方法:无菌条件下采集兔骨髓,肝素抗凝,经淋巴细胞分层液(相对密度为1.077)密度梯度离心分离骨髓单个核细胞,通过贴壁培养法获得骨髓间充质干细胞(bonemarrow-mesenchymalstemcell,MSC),然后在内皮细胞生长环境中进行诱导分化,观察细胞的体外生长特点及其生化特性。结果:MSC每扩增一代,细胞数量增加5～8倍,7.65×103个原代MSCs体外扩增3代即获得1.26×108个细胞。特定条件下贴壁细胞延展呈梭形,形成细胞簇、索状结构及“铺路石”样结构,扩增细胞免疫组化证实表达CD31和VIII因子相关抗原(factorVIIIrelatedantigenvonWille-brandfactor,vWF),具有吞噬乙酰化低密度脂蛋白的功能。结论:MSC扩增能力强,在体外能诱导其向内皮细胞方向分化。     

犬外周血淋巴管内皮祖细胞的分选及其向内皮细胞的诱导分化研究

目的研究犬外周血中CD34^＋／CD133^＋／VEGFR-3^＋淋巴管内皮祖细胞的生物学特征，探讨血管内皮生长因子-C（VEGF-C）／血管内皮生长因子受体3（VEGFR-3）信号途径在淋巴管内皮祖细胞向淋巴管内皮细胞分化中的作用。方法用密度梯度离心法从犬外周血分离单个核细胞，再用流式细胞仪从单个核细胞分选VEGFR-3^＋细胞。通过VEGF-C诱导使VEGFR-3^＋细胞向内皮细胞分化。在扫描电镜和透射电镜下观察细胞表面形态和超微结构，并在共聚焦激光扫描显微镜下观察特征性标志物的表达。结果VEGFR-3^＋细胞同时表达CD34和CD133。经流式细胞仪分析，外周血单个核细胞中CD34^＋／VEGFR-3^＋细胞的含量约为0.13％，VEGFR-3^＋／CD133^＋细胞的含量约为0.08％。CD34^＋／CD^33^＋／VEGFR-3^＋细胞的体积较大，直径约为15μm。经VEGF-C诱导后，细胞数目增多。细胞变为梭形，伸出板状伪足和许多丝状伪足。诱导后1周，细胞表达血管性血友病因子。诱导后2周，细胞表面出现小凹，细胞内可见Weibel-Palade小体。细胞表达淋巴管内皮细胞特异性标志物LYVE-1，CD133标志消失。结论在VEGF-C诱导作用下，外周血中的CD34^＋／CD133^＋／VEGFR-3^＋淋巴管内皮祖细胞能向淋巴管内皮细胞分化。VEGF-C／VEGFR-3信号途径在淋巴管内皮祖细胞的分化过程中起着重要作用。     

苦碟子注射液对动脉粥样硬化症血管内皮细胞功能的干预作用

目的探讨苦碟子注射液对血管内皮细胞分泌功能的保护作用。方法对72例动脉粥样硬化患者在基础药物治疗的基础上,给与苦碟子注射液20 mL静脉滴注,1次.d-1,1周为1个疗程。分别于治疗前及治疗1个疗程后各抽取患者空腹静脉血3 mL,检测血管内皮细胞分泌前列环素(PGI2)、内皮素(ET)及血栓素A2(TXA2)水平,观察治疗前后PGI2、ET、TXA2水平的变化。结果治疗前患者的TXA2、PGI2及ET分别为(29.4±3.5)、(18.9±4.7)及(28.3±4.3)ng.L-1,治疗后分别为(18.7±3.3)、(34.5±3.8)及(17.1±2.1)ng.L-1,治疗后TXA2、ET显著低于治疗前,PGI2显著高于治疗前(均P<0.01)。结论苦碟子注射液能改善和调整血管内皮细胞异常的分泌状态。血管内皮细胞可能是苦碟子作用的靶点之一。     

条带杂交mRNA定量分析及其在血栓调节蛋白基因表达研究中的应用

含血栓调节蛋白cDNA的质粒转染大肠杆菌,经扩增、酶切、凝胶电泳纯化,获得长度为3.7kb的血栓调节蛋白cDNA,后者用随机引物法标记~(32)P,制成血栓调节蛋白的基因探针。将该探针与血栓调节蛋白mRNA进行条带杂交后,分别用β-液闪仪和放射自显影及密度扫描仪作定量分析。用本法研究了血管内皮细胞中血栓调节蛋白基因表达及血小板血栓调节蛋白mRNA的含量。     

Both sides nanopatterned tubular collagen scaffolds as tissue‐engineered vascular grafts

Two major requirements for a tissue‐engineered vessel are the establishment of a continuous endothelium and adequate mechanical properties. In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co‐culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano‐scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the case in natural vessels. Both cell types were observed to proliferate and retain their phenotypes in the co‐culture. Copyright © 2010 John Wiley & Sons, Ltd.     

Pro-inflammatory cytokines mediating senescence of vascular endothelial cells in atherosclerosis

Atherosclerosis (AS) is a chronic inflammatory vascular disease, and aging is a major risk factor. The accumulation of senescent vascular endothelial cells (VECs) often leads to chronic inflammation and oxidative stress and induces endothelial dysfunction, contributing to the occurrence and development of AS. Senescent cells can secrete a variety of pro-inflammatory cytokines to induce the senescence of adjacent cells in a paracrine manner, leading to the transmission of signaling of cellular senescence to neighboring cells and the accumulation of senescent cells. Recent studies have demonstrated that several pro-inflammatory cytokines, including IL-17, TNF-α, and IFN-γ, can induce the senescence of VECs. This review summarizes and focuses on the pro-inflammatory cytokines that often induce the senescence of VECs and the molecular mechanisms of these pro-inflammatory cytokines inducing senescence of VECs. Targeting the senescence of VECs induced by pro-inflammatory cytokines may provide a potential and novel strategy for the prevention and treatment of AS.     

Amorphous nanosilica particles evoke vascular relaxation through PI3K/Akt/eNOS signaling

There have been several reported studies on the distribution and/or toxicity of nanosilica particles. However, the influence of these particles on blood vessels through which they are distributed is poorly understood. Hence, we investigated the effects of nano‐ and micromaterials on blood vessel shrinkage and relaxation. Nanosilica particles with diameters of 70 nm (nSP70) were used as the nanomaterial, and particles of 300 and 1000 nm (nSP300 and mSP1000, respectively) were used as micromaterials. A rat thoracic aorta was used as the test blood vessel. The nano‐ and micromaterials had no effect on vessel shrinkage. Of the nano‐ and micromaterials tested, only nSP70 strongly evoked vascular relaxation. Vascular relaxation evoked by nSP70 was almost completely inhibited by the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin. In addition, the selective nitric oxide synthesis inhibitor NG‐nitro‐l ‐arginine methyl ester, which inhibits endothelial nitric oxide synthase (eNOS) downstream of PI3K signaling, inhibited vascular relaxation evoked by nSP70. In an analysis using bovine aortic endothelial cells (bAECs), nSP70 phosphorylated protein kinase B (AKT) and eNOS acted downstream of PI3K signaling. PI3K inhibition by wortmannin reduced AKT and eNOS phosphorylation. These results demonstrated that 70‐nm amorphous nanosilica particles evoked vascular relaxation through PI3K/Akt/eNOS signaling. Moreover, it was suggested that nanomaterials, in general, control or disrupt vascular function by activating a known signal cascade.     

The suppressive effect of sphingosine 1-phosphate on monocyte-endothelium adhesion may be mediated by the rearrangement of the endothelial integrins α5β1 and αvβ3

BACKGROUND: Sphingosine 1-phosphate (S1P), known to play important roles in vascular biology, is a bioactive lysophospholipid mediator that maintains endothelial integrity via its cell-surface receptors (S1Ps). In this in vitro study, we aimed to examine the role of S1P in monocyte-endothelium adhesion, which is an important event in the pathophysiology of atherosclerosis. METHODS AND RESULTS: S1P pretreatment of human umbilical vein endothelial cells (ECs), but not U937 cells, effectively suppressed U937-EC adhesion independently from the expression of adhesion molecules, namely ICAM-1, VCAM-1, and E-selectin. This S1P-induced suppressive effect was inhibited by the blockage of S1P(1) and S1P(3) receptors and the specific inhibitors of G(i) protein, Src family proteins, phosphatidylinositol 3-kinase, and Rac1, indicating involvement of these key downstream pathways. Moreover, the RGD peptide and antibodies, which neutralize adhesion via alpha(5)beta(1) and alpha(v)beta(3), effectively inhibited U937-EC adhesion with a degree similar to S1P pretreatment. Both an adhesion assay and flow-cytometric analysis demonstrated that U937 cells adhered through integrins alpha(5)beta(1) and alpha(v)beta(3) expressed on the apical surface of monolayer ECs, and S1P shifted the localization of these integrins from the apical surface to the basal surface. CONCLUSIONS: From the present results, we propose that S1P may contribute to the maintenance of vascular integrity and the regulation of atherogenesis through the rearrangement of endothelial integrins.     

Angiogenesis in inflammatory bowel disease

Both ulcerative colitis and Crohn's disease, the two major forms of inflammatory bowel diseases, are recognized, at the moment, as perplexing and challenging clinical entities, in which several molecules and cell types are implicated. Recent molecular evidence proposes the intestinal microvascular remodelling or angiogenesis, as a phenomenon implicated in the pathogenesis of these chronic inflammatory disorders, together with other proposed theories involved in the pathogenesis of inflammatory bowel diseases, such as genetic, microbacterial and immune factors. Intestinal damage is followed by a physiological angiogenesis, but the abnormal expression of pro- and anti-angiogenic molecules and the changes of vascular cell types could reflect a pathological vascular remodelling. Thus, the inflammation may be favoured and maintained by a pathological angiogenesis. A better understanding of the angiogenic process may facilitate the design of more effective therapies for chronic intestinal inflammation.     

Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance

BACKGROUND: We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin-resistant states. MATERIALS AND METHODS: In VSMC derived from human arterioles and from aortas of insulin-sensitive Zucker fa/+rats and insulin-resistant Zucker fa/fa rats incubated with different concentrations of human regular insulin with or without inhibitors of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3-K), mitogen-activated protein kinase (MAPK), nitric oxide synthase (NOS) and guanosine 3',5'cyclic monophosphate(cGMP)-dependent protein kinase (PKG), we measured protein expression (Western blot) and secretion (ELISA) of VEGF. RESULTS: We found that in VSMCs from humans and from insulin-sensitive Zucker fa/+rats, insulin increases VEGF protein expression and secretion, with mechanisms blunted by wortmannin and LY294002 (PI3-K inhibitors), PD98059 (MAPK inhibitor), L-NMMA (NOS inhibitor) and Rp-8pCT-cGMPs (PKG inhibitor). Also the NO donor sodium nitroprusside (SNP) and the cGMP analogue 8-Bromo-cGMP increase VEGF protein expression and secretion, with mechanisms inhibited by wortmannin and PD98059. The insulin effects on VEGF are impaired in VSMCs from Zucker fa/fa rats, which also present a reduced insulin ability to increase NO. CONCLUSIONS: In VSMCs from humans and insulin-sensitive Zucker fa/+rats: (i) insulin increases VEGF protein expression and secretion via both PI3-K and MAPK; (ii) the insulin effects on VEGF are mediated by nitric oxide. The insulin action on both nitric oxide and VEGF is impaired in VSMCs from Zucker fa/fa rats, an animal model of metabolic and vascular insulin-resistance.     

Prospects for intravascular gene therapy

The goal of this review is to provide an overview of gene delivery systems and candidate genes currently being evaluated for genetic strategies in vascular gene therapy. We will discuss treatment strategies that have been shown by in vivo model systems to be efficacious in promoting neovascularization of ischemic tissue or limiting post-interventional restenosis by inhibiting smooth muscle cell proliferation and/or encouraging re-endothelialization. J. Clin. Apheresis 12:140–145, 1997. © 1997 Wiley-Liss, Inc.