L-NAME对人脐静脉内皮细胞VE-cadherin表达的影响

目的　探讨一氧化氮合酶（NOS）抑制剂N-硝基-L-精氨酸甲酯（L-NAME）对人脐静脉内皮细胞（HUVEC）的eNOS、VE-cadherin蛋白表达的影响。 方法 HUVEC传代培养并用CD31特异性抗体进行鉴定,实验分为对照组、0.1 mmol/L L-NAME组、1.0 mmol/L L-NAME组,运用MTT比色法检测L-NAME对HUVEC细胞活性的影响,并采用免疫荧光细胞化学法或Western blot检测实验各组HUVEC细胞的eNOS、VE-cadherin等蛋白的表达,以观察L-NAME对HUVEC的eNOS蛋白、VE-cadherin蛋白的影响。 结果 95%以上传代后的细胞为CD31免疫阳性细胞。MTT比色法结果显示,随着L-NAME的浓度增加,HUVEC的细胞活力值逐渐减弱。免疫荧光细胞化学或Western blot结果显示,与对照组比较,0.1 mmol/L L-NAME组和1.0 mmol/L L-NAME组 HUVEC中eNOS的表达减弱（P<0.01）,并呈剂量依赖性降低;与对照组比较,0.1 mmol/L L-NAME组和1.0 mmol/L L-NAME组 HUVEC中VE-cadherin的表达增强（P<0.01）,呈剂量依赖性增加。 结论 L-NAME能抑制HUVEC 的eNOS表达和细胞活性,并影响细胞粘附连接的主要蛋白-VE-cadherin的表达。     

Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function.     

腺苷抑制脂多糖诱导的人脐静脉内皮细胞凋亡

目的 探讨腺苷抑制脂多糖（LPS）诱导人脐静脉内皮细胞（HUVEC）的凋亡。方法 将培养的HUVEC分成6组：对照组,二甲基亚砜（DMSO）组,LPS组,低、中和高剂量腺苷组,用荧光显微镜动态观察细胞形态结构,用ELISA检测上清液中TNF-α表达量,用流式细胞技术检测各组细胞凋亡。结果 LPS组细胞凋亡率显著升高,TNF-α的表达量也显著升高（p＜0.05）;不同剂量腺苷能显著抑制LPS诱导的TNF-α的表达,显著抑制细胞的凋亡率（p＜0.05）。结论 一定浓度的腺苷能抑制LPS 诱导的HUVEC释放炎症介质、抑制细胞发生凋亡。     

DAPT在ox-LDL损伤HUVECs过程中的细胞保护作用

目的:探究γ-分泌酶抑制剂N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)在氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)损伤人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)模型中的细胞保护作用及其对Notch信号通路的调控。方法:体外培养HUVECs,用oxLDL处理HUVECs构建细胞损伤模型。实验分为对照组、ox-LDL处理组、DAPT处理组和DAPT+ox-LDL处理组。用倒置相差显微镜观察不同处理方法下细胞的形态变化;CCK-8法检测细胞存活率;Western blot法检测蛋白Notch1、Notch4和Jagged1的表达情况。结果:体外培养HUVECs,倒置相差显微镜下发现ox-LDL处理组细胞死亡和碎片增多,经DAPT预处理后,ox-LDL作用造成的细胞损伤死亡较少,细胞碎片较少。通过CCK-8法检测发现ox-LDL处理组细胞存活率降低,DAPT处理组细胞存活率升高,DAPT预处理后ox-LDL造成存活率降低的幅度变小。在ox-LDL作用下,Notch1和Jagged1蛋白表达量降低,Notch4表达量升高;而DAPT作用下Notch1和Jagged1表达量升高,Notch4表达量降低;ox-LDL与DAPT共同作用时蛋白接近正常水平。结论:ox-LDL对HUVECs具有损伤作用;DAPT减轻ox-LDL对HUVECs造成的损伤;DAPT保护HUVECs免受ox-LDL损伤的作用与Notch信号通路有关。     

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.     

流体剪切应力对人脐静脉内皮细胞Pim-1基因表达的影响

目的　探讨流体剪切应力对人脐静脉内皮细胞（HUVECs）中Pim-1基因表达的影响及可能的信号机制。 方法　酶消化法分离健康产妇新鲜脐静脉获取原代HUVECs,应用平行平板流动腔系统给HUVECs加载不同时间（1、4、6 h）和不同大小（0.5、1.5、3.0 Pa）的层流剪切应力,用实时定量RT-PCR检测Pim-1基因的表达水平,用蛋白质印迹法检测p-Akt和p-STAT3的表达水平,利用Wortmannin (PI3K抑制剂)、 Deguelin (Akt抑制剂) 和AG490 (JAK抑制剂) 信号阻断剂探讨信号转导途径。 结果　HUVECs在1.5 Pa 剪切应力作用4 h后Pim-1基因表达明显增加。不同强度的切应力均会刺激Pim-1基因的表达,其中3.0 Pa表达最强。切应力能显著激活p-Akt和p-STAT3的表达。AG490可以明显抑制Pim-1的表达,而Wortmannin和Deguelin增强Pim-1的表达。 结论　流体剪切应力可诱导HUVECs中Pim-1基因表达,其表达量与刺激时间和剪切应力的强度密切相关,这种作用可能通过JAK/STAT3和PI3K/Akt信号通路调节。     

The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules

Modular tissue-engineered constructs are assembled from sub-mm sized cylindrical collagen gel modules which are covered with a surface layer of human umbilical vein endothelial cells (HUVEC). The resulting construct is permeated by a network of interconnected endothelial cell lined channels to facilitate blood perfusion and nutrient delivery. This design strategy relies critically on the endothelial cells' layer behaving in a non-thrombogenic manner on the module surface and the objective here was to characterize this thrombogenicity. HUVEC prolonged clotting times in whole blood-module mixtures, and enabled slightly heparinized whole blood perfusion of an assembled modular construct in vitro with no increase in platelet loss compared to background levels. Flow cytometry and scanning electron microscopy indicated that HUVEC seeded modules reduced platelet activation and deposition but not leukocyte activation, compared to collagen only modules. Plasma recalcification times on non-stimulated HUVEC were longer compared to stimulated HUVEC but not different than that on collagen only module films and were not prolonged by incubation with a tissue factor blocking antibody. Together these data suggest that a functional non-thrombogenic layer of EC was generated on the module surface and that this layer should be sufficient to maintain continuous blood flow through an engineered modular tissue. In/ex vivo studies are warranted to confirm this conclusion.     

金粉蕨素对甲萘醌损伤人脐静脉内皮细胞的保护作用

目的: 探讨金粉蕨素对氧化应激损伤人脐静脉内皮细胞的保护作用。方法: 以甲萘醌损伤人脐静脉内皮细胞,作为氧化损伤模型;采用MTT法,测定内皮细胞的生长抑制率;用硝酸还原酶法测定培养液中NO₂^-／NO₃^-含量;以Westernblot方法检测细胞内皮型一氧化氮合酶(eNOS)活性及小凹蛋白(caveolin-1)表达。结果: 金粉蕨素呈剂量依赖性地降低甲萘醌对内皮细胞的生长抑制率,增加培养液中NO₂^-／NO₃^-含量,保护eNOS活性、阻止caveolin-1表达下降。结论: 金粉蕨素可能通过caveolin-1/eNOS通路介导对抗甲萘醌对内皮细胞的氧化损伤作用。     

用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达

目的：建立稳定表达绿色荧光蛋白(GFP)的人脐静脉血管内皮细胞（HUVECs），研究小干扰RNA(siRNA)对HUVECs中GFP表达的抑制作用。 方法： 用lipofectamine2000将编码GFP的质粒pN3-EGFP转入HUVECs中。用G418筛选并维持已转化pN3-EGFP的HUVEC(HUVEC-GFP)。利用T7 RNA转录试剂盒，制备可抑制GFP基因表达的siRNA（GFPsiRNA）和无关对照的RNA（control siRNA）。用oligofectamine将siRNA转入HUVEC-GFP中。继续培养48 h后，检测HUVEC-GFP中GFP蛋白和mRNA表达水平。 结果： 用G418筛选获得了HUVEC-GFP细胞株，可以观察到GFP的稳定表达。HUVEC-GFP转化siRNA后48 h，GFP的荧光强度明显下降，而对照组的荧光强度无明显下降。半定量RT-PCR检测显示，GFPsiRNA对GFP mRNA表达有较强的抑制作用，抑制率达40%，而control siRNA对GFP mRNA表达水平无明显的抑制作用。 结论： 利用体外转录合成的siRNA能有效地抑制HUVECs中GFP的表达。     

丹参酮ⅡA对高糖诱导人脐静脉内皮细胞凋亡的作用

目的:观察丹参酮ⅡA对高糖刺激人脐静脉内皮细胞凋亡的影响,并探讨相关的作用机制。方法:四甲基偶氮唑蓝(MTT)法检测细胞存活率;流式细胞术Annexin V-FITC/PI双染法定量检测内皮细胞凋亡情况;免疫印迹法检测抗凋亡分子Bcl-2、促凋亡分子Bax以及细胞色素C(cytochrome C,Cyt C)的蛋白表达水平。结果:丹参酮ⅡA可抑制高糖诱导的人脐静脉内皮细胞存活率的减少以及凋亡率的增加。此外,丹参酮ⅡA处理后,Bcl-2蛋白表达显著增加,Bax蛋白表达显著减少,并且线粒体Cyt C释放受到明显抑制。结论:丹参酮ⅡA可以通过调节线粒体凋亡信号通路相关蛋白表达而对抗高糖诱导内皮细胞的凋亡。     

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。     

脂多糖诱导血管内皮细胞凝血途径相关因子基因表达变化

目的：观察脂多糖(LPS)对脐静脉血管内皮细胞(HUVECs)表达组织因子(TF)、组织因子抑制物(TFPI)和凝血酶调节蛋白(TM)的影响。方法：应用胰酶消化HUVECs并进行传代培养，用生长良好的第2、3代细胞进行实验。同时应用CCK-8测定细胞在不同浓度的LPS(1-100 mg/L)处理前后细胞活性；应用反转录聚合酶链反应(RT-PCR)法检测细胞内TM、TF和TFPI mRNA水平。结果：浓度为10 mg/L的LPS对细胞活力与对照组相比没有显著差异。浓度为10 mg/L的LPS作用使HUVECs显著上调TF mRNA表达，6-24 h可以使细胞TFPI mRNA表达下调,以后渐恢复正常表达，72 h达到正常对照组水平，同时下调TM mRNA表达。结论：LPS(10 mg/L)对HUVECs的活性不造成直接的影响，可显著上调HUVECs的TF mRNA转录，抑制TFPI mRNA 的转录，而不改变TM mRNA的转录，这可能与LPS在感染过程中诱导血栓形成,血液凝固和DIC发生相关。     

SPP对人脐静脉内皮EA.hy926细胞表达粘附分子的作用

目的:通过研究在N,N-二甲基鞘氨醇(DMS)作用下,人脐静脉内皮EA.hy926细胞株(EA株)表达粘附分子CD54(ICAM-1)和CD62p(PS)的变化,探讨第二信使1-磷酸鞘氨醇(SPP)参与动脉粥样硬化(AS)粘附过程的机制。方法:采用流式细胞仪分群作用衡量EA细胞株和单核细胞之间的粘附率,用细胞免疫组化法检测DMS作用于EA株后引起的粘附分子的表达。结果:随着DMS作用剂量或作用时间的增加,CD54与CD62p的表达均减少,同时粘附率也相应地下降。结论:AS早期的粘附过程可能是通过SPP来转导信息,以促进粘附分子表达。     

重组可溶性人CD40L诱导人脐静脉内皮细胞损伤

目的:探讨重组可溶性人CD40L(rsh CD40L)对人脐静脉内皮细胞(HUVECs)的损伤作用及其在动脉粥样硬化中的作用。方法:应用rsh CD40L刺激人脐静脉内皮细胞12 h;MTS法观察HUVECs的生存活性,ELISA法测内皮细胞E-选择素(E-selectin)、细胞间黏附分子(ICAM)-1、组织因子(TF)、组织因子途径抑制物(TFPI)表达的变化,比色法测脂质过氧化物丙二醛(MDA)含量及超氧化物歧化酶(SOD)活力。结果:与正常组比较,不同浓度的rsh CD40L(0.5、1、2、3 mg/L)对内皮细胞的生存活性无明显影响;0.5 mg/L rsh CD40L即可增加内皮细胞E-selectin、s ICAM-1、TF、TFPI的分泌,差异有统计学意义(P0.01),同时增加内皮细胞MDA的含量、降低SOD活性(P0.05)。结论:0.5~3 mg/L rsh CD40L对内皮细胞生存活性无明显影响,但已经引起内皮细胞功能障碍,增加内皮细胞炎症和外源性凝血反应,诱导内皮细胞脂质过氧化物损,使其抗氧化能力下降。     

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。     

Three-dimensional cell grafting enhances the angiogenic efficacy of human umbilical vein endothelial cells

Despite the great potential of cell therapy for ischemic disease, poor cell survival after engraftment in ischemic tissue limits its efficacy. Here we tested a hypothesis that three-dimensionally grafted human umbilical vein endothelial cell (HUVEC) spheroids would exhibit improved angiogenic efficacy following transplantation into mouse ischemic limbs compared with HUVECs prepared by conventional two-dimensional monolayer culture. One day after surgical induction of hindlimb ischemia in athymic mice, HUVECs cultured in monolayer or HUVEC spheroids were transplanted intramuscularly into ischemic limbs. Four weeks after the treatment, in the spheroid HUVEC transplantation group, we observed increased hypoxia-inducible factor-1α expression, decreased apoptosis, and increased HUVEC survival in the ischemic tissue compared with the monolayer HUVEC transplantation group. Transplantation of HUVEC spheroids also resulted in enhanced and prolonged secretion of paracrine factors as well as enhanced expression of factors involved in the recruitment of circulating angiogenic progenitor cells. In summary, transplantation of HUVECs as spheroids enhanced cell survival, increased paracrine factor secretion, and showed a potential as a therapeutic method to treat ischemic tissue damages by promoting angiogenesis.