尼古丁促人脐静脉内皮细胞凋亡

目的研究不同浓度尼古丁对人脐静脉内皮细胞的促凋亡作用及作用机制。方法体外培养人脐静脉内皮细胞,根据尼古丁浓度分组为10-6、10-7和10-8mol/L组及对照组。尼古丁作用24 h后,用CCK-8法检测细胞增殖,Annexin V-FITC荧光双染色法检测细胞凋亡。Western blot检测Bax、Bcl-2和PARP-1蛋白表达,对其作用机制进行研究。结果与对照组相比,10-6、10-7mol/L尼古丁组细胞增殖活性下降(P0.05);凋亡数目增多(P0.01)。促凋亡蛋白Bax的表达量增加(P0.01);抑凋亡蛋白Bcl-2表达明显减少(P0.01);PARP-1表达增加(P0.01)。结论一定浓度的尼古丁对血管内皮细胞具有促凋亡作用,加速动脉粥样硬化性疾病的发生发展。     

汉坦病毒诱导人脐静脉内皮细胞HSP70的表达及意义

目的: 研究人脐静脉内皮细胞 (HUVEC)感染汉坦病毒(Hantavirus, HTV)后应激反应的规律及意义。方法: 采用免疫细胞化学染色法和核酸分子原位杂交, 检测热休克蛋白 70(HSP70)的表达; 用RT --PCR观察HSP70mRNA水平变化的规律。结果: HTV感染HUVEC后, 免疫细胞化学染色法可检出HSP70呈高表达, 原位杂交发现细胞质内有HSP7mRNA的阳性信号。感染后不同时间点RT- PCR的结果表明与对照组相比较, HSP70mRNA的水平在感染后迅速升高并持续高表达 (P<0. 05 )。结论: HTV可诱导HUVEC高表达HSP70。HSP70可能具有抑制病毒复制和保护内皮细胞的作用。     

雌激素对人脐静脉内皮细胞HSP27表达的影响

目的：观察人脐静脉内皮细胞（human umbilical vein endothelial cell,HUVEC）中热休克蛋白27（HSP27）表达在雌激素（estrogen,E）诱导下的改变。方法：分别用10–9 M、10–8 M、10–7 M雌二醇（estradiol,E2）以及10–6 M雌激素受体拮抗剂他莫昔芬（tamox ifen）处理HUVEC后,采用Western blot法和RT-PCR法检测HUVEC中HSP27蛋白和m RNA的表达水平。结果：与对照组相比,无论是蛋白水平还是m RNA水平,10–9 mol/L E2对HUVEC中HSP27表达没有明显影响;10–8、10–7 mol/L E2诱导HUVEC中HSP27表达逐渐增加（P〈0.05）;而HUVEC与10–6 M他莫昔芬、10–7 M雌二醇共同孵育,其HSP27水平和单纯10–7 M雌二醇处理相比明显减少（P〈0.05）。结论：外源性E2能诱导HUVEC中HSP27的表达,呈剂量依赖性。他莫昔芬能阻断E2的这种上调HSP27的作用,提示雌激素诱导内皮细胞HSP27的表达依赖ER。     

尼古丁对人脐静脉内皮细胞凋亡及坏死的影响

目的不同浓度尼古丁对人脐静脉内皮细胞（HUVECs）凋亡及坏死的影响。方法用CD34以免疫组织化学法鉴定HUVECs。通过3种不同浓度的尼古丁（3、30、300 ng/ml）刺激HUVECs 24 h后,用流式细胞仪检测其凋亡及坏死率。结果低浓度尼古丁促进细胞凋亡最强,而随着尼古丁浓度的增加,细胞凋亡率反而逐渐降低,各组差异具有统计学意义（P〈0.05）;此外,随着尼古丁浓度增加,细胞坏死率也日益增多,各组差异具有统计学意义（P〈0.05）。细胞坏死率与尼古丁浓度呈正相关（r=0.675）。结论尼古丁对HUVECs凋亡的影响具有浓度依赖性,细胞坏死率与尼古丁浓度呈正相关。     

不同培养基对人脐静脉内皮细胞体外培养效果的影响

目的研究不同培养基对人脐静脉内皮细胞体外培养生长的影响,探讨内皮细胞的最佳体外扩增培养条件。方法取健康产妇分娩后脐带,用胶原酶Ⅰ消化后得脐静脉内皮细胞,进行原代培养,并用免疫荧光染色的方法对细胞进行鉴定,传代培养时则分别采用RPMI-1640培养基和EGM-2培养基,倒置显微镜观察两种培养条件下细胞的生长状态,同时利用流式细胞仪检测其生长周期,对比培养效果。结果 EGM-2组内皮细胞生长良好,2d后贴壁生长的细胞可达90%。EGM-2组S期细胞比例为（29.07±1.48）%,RPMI-1640培养基组S期细胞为（17.58±3.49）%,二者比较差异有统计学意义（P〈0.01）。结论 EGM-2培养基更适合人脐静脉内皮细胞的体外传代扩增培养。     

模拟微重力对人脐静脉内皮细胞血管生成素产生的影响

目的探讨模拟微重力对人脐静脉内皮细胞（HUVEC）生成血管生成素（ANG）的影响。方法培养HUVEC，分为模拟微重力组、地面静止对照组及剪切力对照组，在培养第24h、48h、72h和96h分别收集培养上清液，用ELISA方法检测ANG的浓度变化；固定一部分细胞，应用免疫细胞化学方法检测ANG在HUVEC中的表达情况。结果无论是应用ELISA方法检测上清液中的ANG，还是应用免疫细胞化学的方法检测ANG在细胞内的表达，均发现与地面静止及剪切力对照组相比，模拟微重力组HUVEC的ANG生成增加（P〈0．05）。结论应用回转器模拟微重力能促进体外培养的HUVEC生成ANG。     

登革病毒Ⅱ型对人脐静脉血管内皮细胞通透性的研究

目的 研究登革病毒Ⅱ型(DENV-2)对人脐静脉血管内皮细胞(HUVEC)通透性的影响.方法 DENV-2病毒滴定,DENV-2接种HUVEC单层细胞,用间接免疫荧光法动态观察DENV-2感染HUVEC的情况(30 min,l、3、6、12、24、30、36、42、48、72 h),实时定量荧光PCR方法检测病毒载量,transwell法检测DENV-2对HUVEC通透性的影响,透射电镜观察细胞超微结构的改变.结果 DENV-2对HUVEC通透性的影响30 min时作用最为明显,其次为42 h时.且病毒载量与HUVEC通透性改变呈正相关.透射电镜结果显示有细胞微绒毛脱落,胞质溶解,部分核膜间隙增宽,甚至是细胞核中也存在裂隙,部分线粒体嵴消失甚至空化,线粒体髓样变,包膜不完整.结论 DENV-2对HUVEC通透性有影响,为探究登革病毒的发病机制提供一定的理论依据.

Abstract：

Objective To research Dengue virus type 2 (DENV-2) to human umbilical vein endothelial cells (HUVEC) permeability. Methods The titration of DENV-2 was detected and HUVEC infection DENV-2 layer of cells. At different time points after infection (30 min, 1 h, 3 h, 6 h, 12 h, 30 h,36 h, 24 h, 42 h, 48 h,72 h), the permeability in HUVEC were detected after Dengue virus infection. The virus load in HUVEC was detected by quantitative real-time PCR. And ultrastructure in HUVEC was observed by electron microscope. Results The permeability in HUVEC was changed after Dengue virus infection by time lasting. The most permeability in HUVEC was changed after Dengue virus infection at 30 min and 42 h. And at the same time the virus load were most value in all of time. The results of the cell membrane were changed by electron microscope. The deciduous cell microvillus and dissd endochylema were founded. And some of nuclear membrane blank was wided by Dengue virus. Even the leakage was founded in cell nucleus. The other change included that disappearance mitochondrial and vacuolization crista, elder pith change mitochondria. In addition, the complete of cell membrane were dissolved. Conclusion The permeability of HUVEC was changed by DENV-2. To explore the pathogenesis of Dengue virus provide certain theoretical basis.     

人脐静脉内皮细胞受剪切力作用后原癌基因c-fos和c-myc蛋白的表达

本文的研究目的是揭示不同的流动剪切力对体外培养的人脐静脉内皮细胞c-fos和c-myc蛋白表达的不同影响。我们利用平行板流动小室装置控制剪切力大小和持续时间。然后用免疫组织化学方法对受剪切力作用后的内皮细胞进行染色并进行图像分析。得以一些重要结果。c-fos蛋白表达在静态时非常低，4dynes/cm^2和10dynes/cm^2的剪应力都可诱导c-fos蛋白水平迅速增加，尤其是10dynes/cm^2的剪应力。并随时间的增加而增加，到1.0h达到高峰，随后下降，2.5h时接受基础水平。c-myc蛋白在静态时表达也非常低，经剪切力作用后缓慢增加，c-myc蛋白水平明显较c-fos低，而且剪应力为4dynes/cm^2和10dynes/cm^2两种情况对c-myc蛋白表达的影响基本无差异。其高峰在1.5h。随后下降，在2.5h时接受基础水平。结果表明人脐静脉内皮细胞c-fos蛋白表达具有对剪切力大小和时间的依赖性；c-myc蛋白表达具有时间依赖性。     

外源性精胺对人脐静脉内皮细胞的损伤作用及机制研究

目的： 观察外源性精胺(Sp)对传代培养的人脐静脉内皮细胞(HUVECs)的作用,并初步探讨其可能机制。方法：采用传代培养的方法,观察不同浓度精胺(50 μmol/L-5 mmol/L)对HUVECs作用2 h、4 h的影响。观察指标：(1)倒置显微镜下观察HUVECs形态学变化;(2)透射电镜观察HUVECs超微结构改变;(3)细胞活力测定; (4)MDA含量测定;(5)SOD活性测定。结果：对HUVECs影响的各项观察指标,50 μmol/L Sp与正常对照组比较,无显著差异(P>0.05);200 μmol/L、1 mmol/L、5 mmol/L Sp对HUVECs有损伤作用,作用4 h的细胞损伤较2 h重,其特点为细胞活力降低,超微结构损伤,MDA含量升高,SOD活性降低。 结论：Sp呈浓度依赖性引起HUVECs损伤,其作用机制可能与氧自由基大量产生导致的细胞膜脂质过氧化有关。     

红景天抑制人脐静脉内皮细胞生长的初步研究

目的:利用体外培养人脐静脉内皮细胞,观察中药红景天对细胞生长的影响,初步探讨急、慢性高原病患者服用中药红景天防治高原病及改善症状等的作用机制。方法:培养人脐静脉内皮细胞EVC-304,设对照组与加药组,加药组分别加入不同浓度的红景天,培养3d后计数。加药组及对照组细胞用瑞氏染料染色并拍照。收集细胞以流式细胞术检测细胞周期。结果:对照组细胞形态正常,成梭形,排列紧密,分散均匀。加药组细胞数量明显减少,细胞皱缩,聚集成团,形态各异。流式细胞术检测显示加药组G1期细胞含量增多,S期细胞减少。结论:红景天具有抑制血管内皮细胞生长的作用,可能是通过抑制细胞的增殖来抑制内皮细胞生长。抑制血管内皮细胞生长对于阻止血管内膜增生,防止形成肺动脉高压,降低慢性高原病发病率具有实际应用意义。     

内皮样细胞与脐静脉内皮细胞的比较

BACKGROUND: Stem cells are induced to differentiate into endothelial-like cells that can be used for the treatment of diabetic lower extremity vascular disease. However, it is unclear whether these endothelial-like cells can completely replace endothelial cells to improve vascular disease and what are the differences between endothelial-like cells and endothelial cells. OBJECTIVE: To explore the differences and similarities between endothelial-like cells and human umbilical vein endothelial cells in the aspects of morphology, function, and viability. METHODS: Umbilical cord mesenchymal stem cells and umbilical vein endothelial cells were isolated, cultured and identified using flow cytometry and immunohistochemical method. Isolated umbilical cord mesenchymal stem cells were induced in DMEM-LG/F12 containing 10 µg/L vascular endothelial growth factor, 10 µg/L basic  fibroblast growth factor and 2% fetal bovine serum to differentiate into endothelial-like cells followed by immunohistochemical identification. To compare endothelial-like cells with human umbilical vein endothelial cells, cell migration detection, active substance measurement and three-dimensional angiogenesis test were performed. RESULTS AND CONCLUSION: Isolated umbilical cord mesenchymal stem cells strongly expressed the surface markers of mesenchymal stem cells, and human umbilical vein endothelial cells strongly expressed CD31 and VWF. After induction, the umbilical cord mesenchymal stem cells were identified to highly express CD31 and VWF. Through cell migration, active substance and three-dimensional angiogenesis tests, endothelial-like cells were similar to endothelial cells in the function and activity, and superior to endothelial cells.  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     

Cytotoxicity of tumor necrosis factor-alpha for human umbilical vein endothelial cells

Human umbilical vein endothelial cells were examined for sensitivity to killing by human recombinant tumor necrosis factor-alpha (TNF-alpha). Treatment of the cells with concentrations of TNF-alpha up to 50 ng/ml for 18 hours did not produce evidence of cytotoxicity. However, a marked cytotoxic effect was found when TNF-alpha pretreated cells were incubated in Hanks' balanced salt solution for a further 4 hours. Exposure of the cells to heat-inactivated or antibody-neutralized TNF-alpha did not result in cytotoxicity. Human recombinant interleukin-1 also lysed endothelial cells under the same conditions, whereas human recombinant macrophage-colony stimulating factor did not. Inclusion of superoxide dismutase, catalase, or soybean trypsin inhibitor in the culture medium during the time of endothelial cell exposure to TNF-alpha had no protective effects. Likewise, allopurinol (a xanthine oxidase inhibitor) and nordihydro-guaiaretic acid (a lipoxygenase inhibitor) were not protective under the same conditions. In contrast, the ferric iron chelator deferoxamine mesylate and three different cyclooxygenase inhibitors provided significant protection against TNF-alpha induced cytotoxicity. When human dermal fibroblasts and human squamous epithelial cells were used in place of the umbilical vein endothelial cells, these cells were resistant to TNF-alpha mediated killing. These findings demonstrate that under the experimental conditions employed, TNF-alpha is cytotoxic for human umbilical vein endothelial cells. This may have implications in a number of in vivo situations in which TNF-alpha is thought to play a role.     

A cell kinetic analysis of human umbilical vein endothelial cells

Cultures of normal human cells ‘age’ and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43±0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47±0.3% as assessed by senescence associated β-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31±0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.     

Enterovirus infection and activation of human umbilical vein endothelial cells

Gastrointestinal tract associated lymphoid tissue is considered to be the main replication site for enteroviruses. In order to invade tissues to reach pancreatic islets, cardiac muscles, and other secondary replication sites, the virus has to survive circulation in the blood and find a way to get through endothelial cells. In the present study, the susceptibility of human endothelial cells to infections caused by human parechovirus 1 and several prototype strains of enteroviruses, representing different species (human poliovirus, human enterovirus B and C), and acting through different receptor families was examined. Primary endothelial cells isolated from human umbilical vein by collagenase perfusion and also an established human endothelial cell line, HUVEC, were used. Primary endothelial cells were highly susceptible to several serotypes of enteroviruses (coxsackievirus A13, echoviruses 6, 7, 11, 30, and poliovirus 1). However, coxsackievirus A 9 and echovirus 1 infected only a few individual cells while human parechovirus 1 and coxsackie B viruses did not show evidence of replication in primary endothelial cells. In general, primary endothelial cells were more sensitive to infection-induced cytolytic effect than HUVEC. Activation of endothelial cells by interleukin-1beta did not change the pattern of enterovirus infection. Immunofluorescence stainings of infected primary endothelial cells showed that expression of activation markers, E-selectin, and intercellular adhesion molecule-1, was clearly increased by several virus infections and the former molecule also by exposing cells to UV-light inactivated coxsackieviruses. In contrast, human leukocyte antigen-DR expression was not increased by virus infection.     

脂联素抗人脐静脉内皮细胞氧化损伤的作用研究

目的:研究脂联素（APN）对过氧化氢(H2O2)诱导人脐静脉内皮细胞 (HUVECs) 脂质过氧化的影响。方法：采用Annexin V-FITC标记后流式细胞检测方法观测H2O2引起的细胞凋亡情况。用硫代巴比妥酸微量法测定丙二醛(MDA),黄嘌呤氧化酶法测总超氧化物歧化酶(SOD),可见光法测过氧化氢酶(CAT）。体外自由基捕捉实验检测APN对超氧阴离子和羟自由基的清除率。结果：APN能明显抑制H2O2引起的细胞凋亡。APN预处理HUVECs后,H2O2（200 μmol/L,6 h）所致的细胞膜脂质过氧化反应明显减弱,MDA产生减少,CAT和SOD活性增加,但仅以APN孵育HUVECs对SOD和CAT活性没有明显的影响。结论：结果表明APN对超氧阴离子和羟自由基有明显的清除效果。APN通过抗脂质过氧化发挥抑制细胞凋亡,保护血管内皮细胞抵抗损伤的作用。     

天麻素抑制脂多糖诱导的人脐静脉内皮细胞的自噬

目的 探讨天麻素(Gas)在脂多糖 (LPS) 诱导的人脐静脉内皮细胞 ( HUVECs) 自噬中的作用。方法 用不同浓度的Gas(5、10、20、50μmol/L) 预处理 HUVECs 1h,再加入1mg/L LPS共培养 12 h后,分别提取细胞总蛋白及mRNA,采用 Western blotting 及 RT-PCR 检测 Beclin-1、ATG5 及 LC3的表达变化;采用丹酰尸胺( MDC) 染色检测细胞中自噬小体(autophagosome)的表达变化;采用MTT检测 HUVECs 生存率的变化。 结果 LPS 诱导 HUVECs 产生自噬;Gas 预处理后自噬水平明显降低且呈剂量依赖性;与此同时细胞的生存率明显增加。 结论 LPS诱导 HUVECs 产生自噬;Gas 通过抑制由LPS 诱导产生的自噬,提高 HUVECs 的生存率。     

纳米羟基磷灰石对人脐带静脉血管内皮细胞的毒性评价

背景：有研究应用脉冲激光沉积合成技术在人工心脏机械瓣膜上沉积胶原制备了新型纳米羟基磷灰石薄膜涂层。 目的：观察此种新型纳米羟基磷灰石薄膜对人脐带静脉血管内皮细胞的毒性。 方法：分别采用纳米羟基磷灰石薄膜常温浸提液、纳米羟基磷灰石薄膜高温浸提液、高密度聚乙烯及苯酚溶液培养人脐静脉血管内皮细胞,72 h内倒置相差显微镜下观察细胞生长状况;培养7 d时采用CCK-8法检测细胞增殖与毒性分级。 结果与结论：培养24 h,纳米羟基磷灰石薄膜常温浸提液组、纳米羟基磷灰石薄膜高温浸提液组和高密度聚乙烯组细胞生长良好,呈梭形,折光性强,3组细胞形态、数量无明显差异;苯酚溶液组细胞多为悬浮、圆形、固缩的死细胞;48 h时,除了苯酚溶液组外,其余3组细胞数量明显增加,细胞生长密集,至72 h时细胞生长旺盛,间隙显著减小。培养7 d内,纳米羟基磷灰石薄膜常温浸提液组、纳米羟基磷灰石薄膜高温浸提液组和高密度聚乙烯组细胞增殖活性无差异,均高于苯酚溶液组(P < 0.05),纳米羟基磷灰石薄膜毒性级别为0至1级,表明纳米羟基磷灰石人工心脏机械瓣膜有良好的组织细胞相容性,无毒性作用。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) was immobilized on substrata in photoreactive gelatin to control the adhesion and growth of vascular endothelial cells. The gelatin and VEGF were mixed in water and cast on a polystyrene dish or a silane-coated glass plate. The surface was then photoirradiated in the presence or absence of a photomask and washed. Toughness of the immobilized material was confirmed by ethanol treatment. Human umbilical vein endothelial cells (HUVECs) grew on the immobilized VEGF but not on a nontreated surface. Growth of HUVEC increased significantly with an increase in the amount of immobilized VEGF, and the effects were inhibited by treatment with anti-VEGF antibody. Thus, immobilized VEGF specifically interacted with HUVECs to permit growth in culture. Micropatterning of HUVEC cultures was also achieved using micropattern-immobilized VEGF. This patterning technique may be useful for the formation of blood vessel networks in vitro.