血管内皮生长因子对培养内皮细胞合成一氧化氮的影响

目的 :探讨血管内皮生长因子 （VEGF） /血管渗透因子 （VPF）对内皮细胞 （EC）分泌一氧化氮 （NO）的影响。方法 :将培养的人脐静脉内皮细胞 （HUVEC）随机分为 6组 （n=6 /组 ） :1正常对照组 ;2 VEGF 1ng/ m l;3VEGF 10ng/ m l;4VEGF 10 0 ng/ m l;5低氧组 ;6低氧组 +VEGF 10 0 ng/ m l。采用硝酸还原酶法测定培养液中的 NO2 - ,NO3 -的含量以反映 NO水平。结果 :VEGF促进正常 EC分泌 NO,在一定浓度范围内呈剂量依赖性 ;低氧损伤 EC,使其分泌 NO减少 ;VEGF 10 0 ng/ ml预处理可保护 EC免受低氧损害 ,保持正常分泌 NO的功能。结论 :VEGF能够调节 EC的功能 ,为其促血管形成作用中 EC分裂、增殖、迁移奠定物质基础     

Cyclic strain delays the expression of tissue factor induced by thrombin in human umbilical vein endothelial cells

Most studies of tissue factor (TF) expression in endothelial cells (EC) are performed under stationary culture conditions. The purpose of this study was to determine the influence of mechanical stimuli such as cyclic strain (CS) on the expression of TF in EC exposed to thrombin (Thr). Human umbilical vein endothelial cells (HUVEC) were exposed to 4 U·mL⁻¹ Thr in the presence or absence of 10% average CS at 60 cycles·min⁻¹ and then TF expression was measured. TF messenger RNA (mRNA) expression peaked at 2 hours in HUVEC exposed to Thr, but at 4 hours in HUVEC exposed to both Thr + CS. TF expression was inhibited by p38 and extracellular signal-regulated protein kinase (ERK) inhibitors. For both Thr or Thr + CS stimuli, p38 and ERK activity peaked at 5 minutes (p < 0.05). Nuclear factor-kappa B levels remained high in the Thr group but not in the Thr + CS group, while Egr-1 levels were elevated in the Thr + CS group. We demonstrated CS-delayed, Thr-induced TF mRNA expression in HUVEC, which may be modulated by p38 and ERK inhibitors.     

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

INTRODUCTIONGene therapy is a novel technology leading to improved treatments of some types of cancer[1-3]. On the other hand, anti-angiogenic therapy has also been proven to be a rational approach in the treatment of solid tumors also[4-9]. Selective del…     

缺氧条件下血管活性因子对血管内皮细胞中血管内皮生长因子表达的影响

目的　探讨在缺氧条件下人脐静脉血管内皮细胞血管内皮生长因子 (VEGF)表达及缩血管活性物质内皮素 ,舒血管活性物质NO和NO抑制剂LNNA对VEGF基因表达的影响。方法　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像分析等观察VEGFmRNA和蛋白表达水平。结果　缺氧 6h内皮细胞可见VEGF表达。ET可促进VEGFmRNA的表达 ,NO可明显抑制VEGFmRNA的表达 ,NO抑制剂LNNA也影响VEGFmRNA的表达。ELISA检测VEGF蛋白水平分别为 6h组 (8 2± 1 1) μg/L ,ET +6h组 (9 37± 1 0 2 ) μg/L ,NO +6h组 (2 86± 0 91) μg/L ,LNNA +6h组 (14 75± 1 87)μg/L。 结论　缺氧可诱导人脐静脉血管内皮细胞分泌VEGF并受血管活性物质的调控 ,ET促进其表达 ,NO抑制其表达。     

cDNA microarray analysis of the gene expression profile of VEGF-activated human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF) is one of the most important factors that stimulate angiogenesis and vascular permeability. To clarify the role of VEGF, we analysed a human cDNA chip containing 7267 human genes to identify genes induced by VEGF in human umbilical vein endothelial cells (HUVECs). One hundred thirty-nine cDNAs, including ninety-nine previously known and forty poorly characterized or novel sequences, were increased more than two-fold by VEGF within twenty-four hours of stimulation. Among them, only five are known to regulate angiogenesis: cyclooxygenase 2 (COX2), heparin-binding epidermal growth factor-like growth factor, early growth response 1 (EGR 1), CYR61, and angiopoietin 2. Fifty-three genes induced within the first two hours were thought to be directly induced by VEGF. Of these, Down syndrome candidate region 1 (maximum induction = 6.1-fold) was the most profoundly induced, followed by Mif1 (KIAA0025; 5.5-fold), COX2 (4.7-fold), EGR 3 (3.7-fold), EGR 2 (3.2-fold), bactericidal/permeability-increasing protein (3.1-fold), and CD1B antigen, b polypeptide (3.1-fold). In addition to the genes mentioned above, there were many poorly characterized or novel genes induced by VEGF. Further analysis of these genes may aid in the elucidation of the molecular mechanisms of angiogenesis or vascular permeability stimulated by VEGF. This revised version was published online in June 2006 with corrections to the Cover Date.     

血管内皮生长因子对培养内皮细胞增殖和移行的影响

目的　研究低氧培养心肌细胞分泌出的血管内皮生长因子 (VEGF)蛋白对培养内皮细胞增殖和移行的影响。方法　将培养的人脐静脉内皮细胞 (HUVECs)分为 4组 :正常对照组 ,低氧培养组 ,含VEGF的低氧培养心肌细胞分泌液组 (分泌液组 ) ,分泌液 +低氧培养组。分别采用流式细胞仪、免疫组织化学等方法检测VEGF对HUVECs细胞周期、增殖细胞核抗原表达、氚一亮氨酸掺入量及HUVECs移行的影响。结果　VEGF增加正常培养和低氧培养HUVECs由静止期 DNA合成前期向DNA合成期的转换 ,使DNA合成期及DNA合成后期 分裂期细胞数增加 ,从而促进HUVECs增殖 ,并使常氧和低氧培养HUVECs增殖细胞核抗原的表达、氚一亮氨酸掺入量及移行细胞数目增加。低氧抑制HUVECs的增殖和移行 ,VEGF可拮抗低氧对HUVECs增殖、移行的抑制作用。结论　低氧培养心肌细胞分泌出的VEGF蛋白不仅增加常氧培养HUVECs增殖、移行 ,而且能够拮抗低氧的抑制作用     

bFGF基因转染对血管内皮细胞迁移的影响及机制

目的研究碱性成纤维细胞生长因子（bFGF）基因转染对人脐静脉内皮细胞（HUVECs）迁移能力的影响,并探讨其可能机制。方法通过基因亚克隆构建真核表达载体pcDNA3．1-bFGF,利用脂质体介导将bFGF基因导入HUVECs内,通过RT-PCR和ELISA法检测基因的表达;bFGF基因转染后的HUVECs通过Transwell小室法检测其迁移能力的变化;Western blot法检测转染后的细胞Raf-1、细胞外调节蛋白激酶2（ERK2）和黏着斑激酶（FAK）蛋白表达变化。结果成功构建了表达载体pcDNA3．1-bFGF,该载体转染HUVECs后,bFGFmRNA和蛋白均显著增加。转染bFGF基因可使HUVECs的体外迁移能力增强,上调ERK2和FAK蛋白的表达,但对Raf-1蛋白表达无影响。结论bFGF基因转染能促进血管内皮细胞的迁移,其作用机制可能与上调ERK2和FAK蛋白表达有关。     

神经生长因子对人脐静脉内皮细胞在体外形成管腔样结构的影响

目的探讨神经生长因子(NGF)对人脐静脉内皮细胞(HUVECs)在体外形成管腔样结构(LLSs)的影响及其信号机制。方法将接种在基质胶上的HUVECs分为空白对照组、NGF组、NGF中和抗体组、对照IgG组、NGF中和抗体+NGF组、对照IgG+NGF组,以相差显微镜直接观察LLSs形态,并通过二脒基苯基吲哚/鬼笔环肽免疫荧光染色观察LLSs的细胞组成。为证实NGF促LLSs形成的信号通路,向培养液中分别预先加入丝裂原活化蛋白激酶3个主要信号通路的特异性抑制剂(PD98059、SB203580及SP600125),观察上述3个信号通路对NGF促LLSs形成的影响,并应用Western blot检测NGF及c-Jun N末端激酶(JNK)特异性抑制剂SP600125对HUVECs JNK表达及活性的影响。结果 NGF组LLSs的平均面积较空白对照组明显增加(P<0.01)。NGF中和抗体+NGF组LLSs的平均面积较对照IgG+NGF组明显减少(P<0.01);NGF中和抗体组LLSs的平均面积较对照IgG组亦明显减少(P<0.01)。NGF可激活HUVECs的JNK信号通路,SP600125可显著减少NGF诱导的LLSs形成(P<0.01)。PD98059及SB203580对NGF诱导的LLSs形成均无明显影响(P>0.05)。结论NGF能促进HUVECs在体外形成LLSs,可能与激活JNK信号通路有关。     

Effects of antioxidants on homocysteine thiolactone-induced apoptosis in human umbilical vein endothelial cells

Background and objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine thiolactone (HcyT), one of the homocysteine metabolites in vivo, is toxic both in vivo and in vitro. The aim of this study was to investigate the effect of HcyT on apoptotic damage in human umbilical vein endothelial cells (HUVECs) and the role of antioxidants in the reduction of HcyT-induced apoptosis. Methods HUVECs were cultured in DMEM supplemented with 20% heat inactivated fetal bovine serum cell cultures were maintained in a humidified 5% CO2 atmosphere at 37℃. Cytotoxicity was determined by MTT assay, which consists of hypodiploid cells with propidium iodide labeling and intracellular reactive oxygen species levels using 2',7'-dichlorofluorescein diacetate as the probe by flow cytometry. Results HcyT (250-2000μM) induced HUVECs apoptosis in a time- and concentration-dependent manner. Reactive oxygen species levels rose in response to increasing HcyT concentrations at 24-h incubation. The reduction of cell apoptosis by N-acetylcysteine, vitamin E, or pyrrolidine dithiocarbamate, occurred simultaneously with a significant decrease in intracellular reactive oxygen species levels. Conclusion HcyT exerts its cytotoxic effects on endothelial cells through an apoptotic mechanism involving cellular reactive oxygen species production. The capacity of N-acetylcysteine, vitamin E, and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular reactive oxygen species correlates well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicates HcyT-generated hydrogen peroxide may provoke cell apoptosis via activating nuclear factor-kappa binding protein.     

Effects of humic acid on the viability and coagulant properties of human umbilical vein endothelial cells

We have previously shown that humic acid (well-water humic acid, HA, and synthetic humic acid, SHA) enhances cell surface expression of tissue factor (TF). Here we report that incubation of human umbilical vein endothelial cells (HUVEC) for 2 hr with HA or SHA cause a rapid rise in TF mRNA levels, as shown by Northern blot analysis. To understand the cytotoxic and fibrinolytic effects of HA and SHA on cultured HUVEC, the cells were treated with varying concentrations of HA and SHA for various periods of time. Both HA and SHA (10–200 μg/ml) inhibited the viability of subconfluent HUVEC, cultured in the presence or absence of 20% FBS (Fetal Bovine serum) in the culture medium, in a dose-dependent manner. Both HA and SHA induced surface changes in the HUVEC as revealed by scanning electron micrography (SEM). However, protocatechuic acid, the monomer of SHA, did not significantly inhibit cell growth, and showed a cytotoxic effect only at 200 μg/ml. Furthermore both HA and SHA stimulated HUVEC to produce plasminogen activator inhibitor (PAI-1) and tissue plasminogen activator (t-PA) in a dose and time dependent fashion; the amount of PAI-1 produced was found to exceed that of t-PA. The monomer of SHA did not have this stimulatory effect. These results distinctly suggest that in addition to the inhibition of viability HA is involved in TF induction and PAI-1 synthesis in HUVEC and these may be some of the plausible mechanisms underlying the thrombotic disorders in Blackfoot disease. © 1996 Wiley-Liss, Inc.     

沙利度胺对人脐静脉内皮细胞增殖和血管生成的影响

目的 探讨沙利度胺治疗血管发育不良所致消化道出血的机制.方法 体外培养人脐静脉内皮细胞至对数生长期,分为空白对照组、溶剂对照组(二甲基亚砜)和不同浓度(10、20、40、60、80、100μg/ml)沙利度胺组,根据加或不加成纤维细胞生长因子(bFGF,10 ng/ml),共分为16组.刺激72 h后,MTT法检测细胞增殖情况,酶联免疫吸附法和实时定量PCR法测定血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)表达.结果 加或不加bFGF刺激,中、高浓度(≥40/μg/ml)沙利度胺均能抑制人脐静脉内皮细胞增殖.未加bFGF刺激时.20μg/ml沙利度胺能明显抑制VEGF表达.加bFGF刺激时,10 μg/ml沙利度胺即能明显抑制VEGF表达.未检出TNF-α表达.结论 体外实验中,沙利度胺能抑制人脐静脉内皮细胞增殖和VEGF表达,从而抑制血管生成,达到治疗血管发育不良所致消化道出血的目的 .     

Effect and mechanism of melatonin's action on the proliferation of human umbilical vein endothelial cells

Melatonin is the major secretory product of the pineal gland and is considered an important natural oncostatic agent. The anticancer activity of melatonin is due to its immunomodulatory, anti-proliferative and antioxidative effects. At present there are no direct data available as to melatonin's possible influence on angiogenesis, which is a major biological mechanism responsible for tumor growth and dissemination. The current study investigated the influence of melatonin on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured, identified, and purified. Cell growth and viability, DNA fragmentation and cell cycle analyses were determined. To elucidate the mechanism of action of melatonin, Western blot analyses for P53, Bax and Bcl-2 expression were carried out. The results demonstrate the anti-proliferative and apoptosis-inducing effects of melatonin; these changes were associated with cell cycle arrest, upregulation of P53 and Bax and downregulation of Bcl-2. Taken together, our data showed that melatonin in high concentrations markedly reduces HUVECs proliferation, induces cellular apoptosis, and modulates cell cycle length. P53 and Bax/Bcl-2 expression changes may be involved in these actions of melatonin.     

Retroviral-mediated gene transfer into human myeloma cells

SUMMARY. SW-480 cells, derived from a primary human colon adenocarcinoma, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma. SW-480 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E-64 (10 μM) but was limited by cell pretreatment with phospholipase A₂. SW-480 cell suspension caused marked dose-dependent decreases in plasma recalcification times using normal, factor VIII-deficient and factor IX-deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW-480 cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW-480 TCIPA. Taken together, these data suggest that SW-480 TCIPA arises from SW-480 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW-480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotien IIb/IIIa and Ib prevent SW-480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC₅₀ 0.09 μM) and rhodostomin (IC₅₀ 0.03 μM) were about 6000 and 18000 times, respectively, more potent than GRGDS (IC₅₀ 0.56mM).     

A20基因抑制内皮细胞组织因子表达的研究

目的　探讨外源性A2 0基因在内皮细胞获得表达及对脂多糖诱导的内皮细胞组织因子表达的影响。方法　DOTAP脂质体介导的pCAGGSEHA2 0和pMAMneo基因转染 ,经G4 18筛选阳性单克隆 ,再用免疫荧光组织化学检测A2 0基因的表达 ,将转染A2 0基因的内皮细胞与未转染的内皮细胞分别加脂多糖 ,经原位杂交及免疫荧光组织化学检测组织因子基因的转录和表达 ,采用裂解内皮细胞一步复钙凝块时间测定法测定组织因子促凝活性。结果　成功构建了pCAGGSEHA2 0真核表达重组体 ,A2 0基因在经G4 18筛选后的内皮细胞中得到有效表达 ,能抑制 70 %组织因子的表达及凝块的形成。结论　A2 0基因能够明显抑制脂多糖诱导的内皮细胞组织因子的表达 ,在心血管血栓形成的预防和治疗中有重要作用     

腺苷对人静脉内皮细胞增殖的影响

目的探讨腺苷对人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）增殖的影响。方法MTT法观察腺苷的最佳作用浓度及最佳作用时问。结果4％FBS浓度下，腺苷具有明显的促HUVEC增殖作用，为最佳浓度。在10-^4 mol／L腺苷终浓度对细胞增殖有明显的促进作用，为最佳腺苷浓度。腺苷的促生艮作用在24h即出现，在48h已达到较高水平。结论在血供基本被阻断或血流基本正常的情况下，腺苷可能对细胞增殖尤影响；但在m流减少的情况下，腺苷的应用可以促进内皮细胞增殖，可能对血管新生有启动作用。     

亚砷酸钠对人脐静脉血管内皮细胞的毒性效应

目的探讨亚砷酸钠对体外培养的人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)毒性效应的分子机制。方法通过观察细胞形态和细胞增殖实验分别观察亚砷酸钠对细胞形态及增殖能力的影响;PI染色结合流式细胞仪检测细胞周期的变化;应用实时定量RT-PCR技术检测c-Jun、c-Myc的mRNA表达水平。结果随着亚砷酸钠浓度增加,漂浮的细胞越来越多;细胞增殖抑制率越来越高;细胞周期结果显示,S期的细胞数明显减少,大多数细胞被阻滞在G0/G1期,呈典型的剂量-效应关系;亚砷酸钠下调HUVEC的c-Myc mRNAs水平。结论亚砷酸钠通过下调c-Myc基因表达,抑制细胞增殖能力,提示其在地砷病引起的血管损伤中发挥重要作用。     

糖基化终产物对人脐静脉内皮细胞血管内皮生长因子mRNA及蛋白表达的影响

目的研究糖基化终产物（AGEs）对人脐静脉山皮细胞血管内皮生长凼子（VEGF）mRNA及蛋白表达的影响，探讨AGEs在糖尿病动脉粥样硬化中的作用。方法将人脐静脉内皮细胞株ECV304用BSA及小同浓度的AGEs孵育24h及400mg／L的AGEs孵育0、12、24及36h，采用原位杂交及Westem blot方法检测VEGFmRNA及蛋白的表达。结果人脐静脉内皮细胞在100、200及400mg／LAGEs孵育24h后，VEGFmRNA及蛋白表达均显著高于BSA对照组（P〈0．05），在用400mg／LAGEs分别孵育12．24及36h后，各组内皮细胞VEGF mRNA及蛋白表达量也明显高于0h对照组（P〈O．05）。结论AGEs可增加人脐静脉内皮细胞VEGF mRNA及蛋白的表达，且与浓度和时间呈正相关。