人脐静脉血管内皮细胞的分离、培养、鉴定及试验研究

血管内皮细胞体外培养,在血管再生、新血管生成机理、内皮细胞在心血管系统疾病中的作用、内皮细胞与造血的关系等方面的研究中有很高的应用价值。本研究借鉴了前人的工作,建立了胶原酶灌流消化获得人脐静脉血管内皮细胞并进行体外培养的方法,探讨了影响人脐静脉血管内皮细胞（HUVEC）分离培养的影响因素。同时应用建立的HUVEC体外培养进行了内皮抑素的抑制活性检测,证明了内皮抑素对于激活增殖的HUVEC的抑制作用,为今后的试验研究打下了基础。     

人脐静脉内皮细胞体外培养的影响因素

脐静脉内皮细胞的体外培养,为深入研究该类细胞学特性,探讨与之有关的众多基础医学课题提供了较为理想的实验手段。其体外培养主要受脐带离体时间、消化酶、培养液、血清、血管内皮细胞生长因子(ECGF)、传代、二氧化碳浓度等影响。     

人脐带动,静脉内皮细胞的异质性研究

异质性是内皮细胞重要的生物学特征之一。本文从体外培养细胞的形态、免疫细胞化学反应和脱落细胞形态等方面研究了人脐带动脉内皮细胞（ＨＵＡＥＣ）和静脉内皮细胞（ＨＵＶＥＣ）的异质性。实验结果表明：１．体外培养的ＨＵＡＥＣ以多角形为主,ＨＵＶＥＣ以长３梭形为主;２．ＨＵＡＥＣ表面的ⅧＲ：Ａｇ较少,ＨＵＶＥＣ表面的ⅧＲ：Ａｇ较多;３．脱落的ＨＵＡＥＣ长而曲折,呈带形,但ＨＵＶＥＣ则短而平直,呈梭形。上述结果     

组织工程静脉血管种子细胞的培养

目的获取可应用于静脉血管组织工程的种子细胞内皮细胞。方法选取北京地区雄性杂种犬,取其双侧颈外静脉,采用翻转后酶消法,获取原代内皮细胞,对其进行原代培养和传代培养,冻存、复苏和鉴定,并利用光学显微镜进行观察。结果获取犬的颈外静脉后,实验采取了翻转后酶消法,所获得的内皮细胞纯度和数量得到了提高;经过鉴定确实为内皮细胞来源;活性好,增殖快,在较短的时间内能够达到后期实验所需数量。结论经体外培养的犬的颈外静脉内皮细胞可以作为组织工程血管的种子细胞。     

胃癌血管靶向肽GX2 对胃癌血管内皮细胞靶向性的鉴定

目的:证明胃癌血管靶向肽GX2能够与胃癌血管内皮细胞特异性结合,在体内对胃癌血管具有靶向性。方法:体外实验,合成GX2与FITC的复合物FITC-GX2,分离原代人脐静脉内皮细胞HUVEC,将HUVEC与人胃癌细胞系SGC7901共培养,建立共培养血管内皮细胞模型来模拟胃癌血管,利用免疫荧光的方法观察GX2与共培养内皮细胞Co-HUVEC的结合情况;体内实验,构建99mTc标记的GX2分子示踪探针,SPECT显像观察GX2的胃癌血管靶向性。结果:免疫荧光结果显示GX2能与Co-HUVEC特异性结合,而与人胃癌细胞SGC7901不结合;SPECT显像结果证明了GX2在荷瘤裸鼠体内能够浓集到肿瘤部位,具有良好的靶向性。结论:GX2能与胃癌血管内皮细胞特异性结合,且具有在体靶向到胃癌血管的能力;GX2具有胃癌诊断的潜在价值,并能应用于胃癌血管抑制治疗。     

离心法与细胞显微操作法对内皮细胞-白细胞黏附特性的初步研究

将哮喘病理血清与在体外培养的大鼠肺内皮细胞共同孵育,分别用离心法和显微操纵技术对细胞黏附进行体外研究。离心结果显示,病理血清刺激下的内皮细胞比正常情况更易与白细胞发生黏附。离心力高到一定程度可以解离部分黏附。从进一步的细胞显微操纵实验看病理血清刺激内皮细胞的功能状态,影响内皮细胞与白细胞发生黏附     

γ分泌酶抑制剂对肝血窦内皮细胞的影响

目的:在建立高纯度小鼠肝血窦内皮细胞的体外培养的基础上研究γ分泌酶抑制剂(DAPT)对肝血窦内皮细胞活性的影响.方法:首先通过胶原酶灌注消化、percoll梯度离心和选择性贴壁分离得到高纯度、可在体外条件下培养的肝血窦内皮细胞,其次用不同浓度的DAPT(15 μmol/L、45 μmol/L、75 μmol/L)处理细胞,然后通过MTT检测细胞增殖情况、Real time PCR检测相关分子改变.结果:在体外条件下DAPT对肝血窦内皮细胞的增殖起到促进作用,这种促进作用随着DAPT浓度的增加相应的增加;DAPT能够导致肝血窦内皮细胞Notch信号下游分子Hes1表达下调,VEGF信号中VEGFR1表达下调,VEGFR2表达上调.结论:γ分泌酶抑制剂(DAPT)通过抑制肝血窦内皮细胞Notch信号,引起肝血窦内皮细胞表面VEGFR1表达下调,VEGFR2 表达上调显著增加肝血窦内皮细胞的活性.     

内皮细胞-血小板血栓体外模型的建立及分析

在内皮细胞培养技术、荧光显微技术和计算机图象处理技术的基础上,建立了内皮细胞－血小板血栓体外动力学模型及其计算机定量分析系统。通过锥板血流模拟装置（Ｃｏｎｅ－ＰｌａｔｅＳｙｓｔｅｍ）结合光－色素法在体外产生血小板血栓,并利用计算机图像处理技术,研究分析了不同剪切应力作用下,内皮细胞的形态变化及血小板吸附情况,定量分析血小板和内皮细胞之间相互作用。该实验模型及其定量分析系统的建立,为深入研究血栓形成和动脉粥样硬化提供了一套在细胞及分子层次作用机理的研究方法,并可进行微量、快速、动态的抗血栓药物的筛选     

缺氧条件下血管活性因子对血管内皮细胞中VEGF表达的影响

 探讨在缺氧条件下人脐静脉血管内皮细胞对血管内皮生长因子 (vascular endothelialgrowth factor,VEGF)表达及缩血管活性物质内皮素 (ET)、舒血管活性物质一氧化氮 (NO)和 NO抑制剂 LNNA对 VEGF基因表达的影响 .体外培养人脐静脉血管内皮细胞 ,经缺氧及血管活性物质处理 .Northern杂交、酶联免疫检测和计算机图象分析等观察 VEGF m RNA和蛋白表达水平 .发现缺氧 6h内皮细胞可见 VEGF表达 .ET可促进 VEGF m RNA的表达 ,NO可明显抑制 VEGFm RNA的表达 ,NO抑制剂 LNNA也影响 VEGF m RNA的表达 .ELISA检测 VEGF蛋白水平分别为 6h组 8.2± 1 .1 ng/ L,ET+6h组 9.37± 1 .0 2 ng/ L,NO+6h组 2 .86± 0 .91 ng/ L,L - NNA+6h组 1 4.75± 1 .87ng/ L.缺氧可诱导人脐静脉血管内皮细胞分泌 VEGF并受血管活性物质ET和 NO的调控 ,ET促进其表达 ,NO抑制其表达 .     

NO供体（SIN—1）损伤内皮细胞的实验研究

选择体外培养人脐静脉内皮细胞(HU-VEC)为研究对象,研究了不同浓度NO供体 SIN-1(3-morpholinosydnonimine)对内皮细胞的作用以及SOD、CAT对内皮细胞的保护作用。结果提示:高浓度SIN-1可严重损伤内皮细胞;SOD、CAT能协同减轻NO对内皮细胞的损伤,说明ONOO~-的产生可能是NO损伤内皮细胞的重要机制。     

Varicose veins as a source of adult human endothelial cells

Endothelial cells can be harvested from segments of adult human saphenous vein in a varicose condition removed from patients having single or bilateral vein ligation and stripping. The cells are harvested by scraping with a scalpel, seeded on to gelatin coated or Primaria flasks and are passaged by removal with a rubber policeman. The cells cultured in this manner are maintained in a growth medium that is not supplemented with growth factors. The cells grow with a cobblestone monolayer morphology, possess angiotensin converting enzyme activity and react with antibodies to Factor VIII antigen. The cells fluoresce brightly after reaction with monoclonal antibodies specific for human endothelial cells. Thus, stripped varicose vein segments provide a readily available source of endothelial cells.     

Comparison of tissue-cultured bovine endothelial cells from aorta and sphenous vein

Summary Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial. This work was supproted by Grants HL-1330 and HL-17269 from NIH.     

Recombinant human laminin-10 (alpha5beta1gamma1). Production, purification, and migration-promoting activity on vascular endothelial cells

The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.     

Comparison of tissue-cultured bovine endothelial cells from aorta and saphenous vein

Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.     

Flow-dependent increase of ICAM-1 on saphenous vein endothelium is sensitive to apamin

The potassium channel blocker tetraethylammonium blocks the flow-induced increase in endothelial ICAM-1. We have investigated the subtype of potassium channel that modulates flow-induced increased expression of ICAM-1 on saphenous vein endothelium. Cultured human saphenous vein endothelial cells (HSVECs) or intact saphenous veins were perfused at fixed low and high flows in a laminar shear chamber or flow rig, respectively, in the presence or absence of potassium channel blockers. Expression of K(+) channels and endothelial ICAM-1 was measured by real-time polymerase chain reaction and/or immunoassays. In HSVECs, the application of 0.8 N/m(2) (8 dyn/cm(2)) shear stress resulted in a two- to fourfold increase in cellular ICAM-1 within 6 h (P < 0.001). In intact vein a similar shear stress, with pulsatile arterial pressure, resulted in a twofold increase in endothelial ICAM-1/CD31 staining area within 1.5 h (P < 0.001). Both increases in ICAM-1 were blocked by inclusion of 100 nM apamin in the vein perfusate, whereas other K(+) channel blockers were less effective. Two subtypes of small conductance Ca(2+)-activated K(+) channel (selectively blocked by apamin) were expressed in HSVECs and vein endothelium (SK3>SK2). Apamin blocked the upregulation of ICAM-1 on saphenous vein endothelium in response to increased flow to implicate small conductance Ca(2+)-activated K(+) channels in shear stress/flow-mediated signaling pathways.     

A novel alternative placement site and technique for totally implantable vascular access ports in non‐human primates

Background Two novel approaches to implanting a central venous catheter port in non‐human primates (NHPs) using peripheral insertion are presented and compared. Methods Sixty vascular access port (VAP) implants were attempted in 52 NHPs by saphenous vein puncture (n = 20) or saphenous vein cutdown (n = 40). Results Fifty eight procedures were successful. Eighteen of 20 VAPs were successfully placed using saphenous vein puncture, and 40 of 40 using saphenous vein cutdown. There were no significant differences between procedures. Mean implantation times were similar between groups. At explant or study endpoint, all 58 VAPs were patent. Conclusions Vascular access port implantation by saphenous vein puncture or saphenous vein cutdown is safe and effective in NHPs. It is less invasive than conventional procedures, has fewer complications, provides outstanding patency, and reduces surgery time. Furthermore, it allows for cooperative in‐homecage VAP use, minimizing handling stress. We recommend these refined methods for long‐term vascular access in NHPs.     

Smoothelin and intermediate filament proteins in human aortocoronary saphenous vein by-pass grafts

The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as smoothelin in the native great saphenous vein. When desmin and smoothelin were present in the neointima, smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.     

Smoothelin and Intermediate Filament Proteins in Humanaortocoronary Saphenous Vein By-pass Grafts

The aim of this immunohistochemical investigation was to study the distribution of the novel cytoskeletal protein smoothelin and the intermediate filament proteins vimentin and desmin in normal human great saphenous vein and in human aortocoronary by-pass vein grafts. Smoothelin was present in most smooth muscle cells in the media of the native vein. In the neointima of the vein grafts that had been in situ for three months or more, smoothelin was, in general, present only in few smooth muscle cells. Desmin was distributed in the same pattern as smoothelin in the native great saphenous vein. When desmin and smoothelin were present in the neointima, smoothelin was detected in more cells than desmin. Vimentin was present in most cells in all wall layers of both the native saphenous vein and the vein grafts. Vascular smooth muscle cells containing vimentin but not desmin or smoothelin are the principal cells in the neointima of human aortocoronary vein grafts. In some grafts, however, all three cytoskeletal proteins were detected in the neointima. The distribution of smoothelin and desmin in aortocoronary vein grafts support the postulate that these proteins are expressed mainly in the contractile smooth muscle cell phenotype.     

Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 micrograms/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.     

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.