树突状细胞的补体攻膜复合物C5b-9模型的建立

目的 建立树突状细胞(DC)的补体攻膜复合物C5b-9模型,为研究补体攻膜复合物对DC的影响奠定基础.方法 诱导人外周血单核细胞分化成为不成熟DC,C5b-9体外组装,激光共聚焦检测C5b-9在DC表面组装情况.结果 成功诱导出不成熟DC,激光共聚焦显示C5b-9完整组装于DC表面.结论 本研究成功建立了DC的补体攻膜复合物C5b-9模型,为将来进一步探讨C5b-9对DC的功能影响奠定了基础.     

血府逐瘀汤促进人脐静脉内皮细胞迁移模型的建立

目的:建立血府逐瘀汤促进人脐静脉内皮细胞HUVEC-12迁移的模型。方法:采用Boyden小室法,将不同数量的细胞置入Boyden小室上室,分别培养4、8、12h。迁移膜经固定、苏木素染色后,于显微镜下观察并计数迁移至膜下室面的细胞。结果:Boyden小室上室中加入2×104个细胞,迁移8h建立的血府逐瘀汤促进HUVEC-12迁移模型为最佳。     

齐墩果酸对氧化损伤模型人脐静脉内皮细胞的保护作用

目的:研究齐墩果酸对氧化损伤模型人脐静脉内皮细胞(HUVECs)的保护作用。方法:分别以含10%胎牛血清DMEM高糖培养液[含氧化低密度脂蛋白(ox-LDL,质量浓度分别为0、25、50、100、200、400μg/ml)]培养HUVECs,CCK-8法检测细胞活性以筛选复制模型最适质量浓度。以0、10、20、40、60、80、100μmol/L齐墩果酸培养HUVECs,CCK-8法检测细胞活性以考察齐墩果酸试验最适浓度。以5、10、20、40μmol/L齐墩果酸作用于氧化损伤模型HUVECs(100μg/ml ox-LDL诱导),CCK-8法检测细胞活性,测定一氧化氮(NO)、一氧化氮合酶(NOS)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)水平。结果:复制模型ox-LDL最适质量浓度为100μg/ml;齐墩果酸试验最适浓度范围为0⁴0μmol/L;5、10、20、40μmol/L齐墩果酸可增加氧化损伤模型HUVECs存活率,增强CAT、GSH-PX、NOS活性,增加NO含量。结论:齐墩果酸能够保护ox-LDL氧化损伤的HUVECs,其保护机制与增强CAT、GSH-PX、NOS抗氧化酶活性有关。     

人脐静脉内皮细胞Caveolin-1基因沉默模型的建立

目的:利用小干扰RNA(siRNA)技术建立人脐静脉内皮细胞(ECV304)小凹蛋白(Caveolin-1)基因沉默模型,为研究Caveolin-1基因在人脐静脉内皮细胞中的作用.方法:通过Ambion专用软件对Caveolin-1基因编码区分析,设计合成短发夹RNA(shRNA)单链.通过T4连接酶将退火反应合成的shRNA双链克隆入含RNA PollII聚合酶表达元件的pENTRTM/U6入门载体,并利用Gateway技术构建相应的慢病毒RNA干扰(RNAi)表达载体.用REAL-TIME RT-PCR、蛋白印迹法分析沉默效率.结果:测序结果显示pENTRTM/U6载体插入的Cavelin-1基因shRNA序列大小及阅读框架正确.LR重组反应后成功构建了针对Caveolin-1基因的RNAi慢病毒表达系统.转染ECV-304细胞获得对Caveolin-1的沉默效率大于85%.结论:成功地构建了针对Caveolin-1基因的RNAi慢病毒表达系统,获得了长期稳定的基因剔除效应.人脐静脉内皮细胞Caveolin-1基因沉默模型能长期保种及传代,为进一步研究Cav-eolin-1基因的功能奠定了基础.     

番红花酸对人脐静脉内皮细胞VCAM-1表达和单核-内皮细胞黏附的影响

番红花酸为天然类胡萝卜素类化合物,具有抗氧化、抗动脉粥样硬化(atherosclerosis,AS)等多种药理作用,但其抗AS作用机制未明。本研究采用体外培养人脐静脉内皮细胞,观察番红花酸(0.1、1、10μmol·L-1)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ,0.1μmol·L-1)诱导的血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)表达和单核内皮细胞黏附的影响,并观察番红花酸对核转录因子κB(nuclear factor kappa B,NF-κB)活化的影响。结果显示,番红花酸可明显减少AngⅡ诱导的VCAM-1表达及单核-内皮细胞黏附(P<0.05,P<0.01),降低细胞内活性氧(reactive oxygen species,ROS)水平和NF-κB活化水平(P<0.05,P<0.01)。番红花酸还可明显增强内皮细胞总抗氧化能力(P<0.01)和超氧化物歧化酶活性(P<0.05,P<0.01)。这些结果提示,番红花酸可有效抑制VCAM-1表达和单核-内皮细胞黏附,其机制可能与番红花酸增强内皮细胞抗氧化能力、减少细胞内ROS水平,进而抑制NF-κB活化有关。     

补体C5b-9复合物对人视网膜色素上皮细胞VEGF表达的影响

目的:观察补体C5b-9复合物对人视网膜色素上皮(RPE)细胞血管内皮生长因子(VEGF)表达的影响.方法:采用大鼠血清接触兔红细胞提取补体C5b-9复合物,提纯、鉴定后分别作用体外培养的人RPE细胞.光学显微镜下观察不同浓度补体C5b-9复合物对细胞的作用效果选取实验剂量.半定量RT-PCR法测量RPE细胞VEGF表达的变化.结果:补体C5b-9复合物刺激浓度>20mg/L,光学显微镜示REP细胞结构破坏;该浓度≤20mg/L,光学显微镜未见REP细胞结构明显改变,选取20 mg/L补体C5b-9复合物刺激RPE细胞4 h,VEGF mRNA表达水平显著增高(P<0.01).结论:补体C5b-9复合物可诱导人RPE细胞VEGF表达上调,可能是补体C5b-9复合物参与脉络膜新生血管(CNV)形成的机制之一.     

姜黄素减弱TNF-α刺激的人脐静脉内皮细胞的炎症反应

炎症和内皮细胞机能障碍是动脉粥样硬化的关键早期事件。研究了姜黄素对肿瘤坏死因子(TNF)-α刺激的人脐静脉内皮细胞(HUVEC)的保护作用,以了解姜黄素对内皮细胞的抗炎作用的途径,为防治细胞因子诱导的内皮细胞机能障碍提供新方法。以 MTT 法检测了姜黄素对 HUVEC 的细胞毒     

麝香及其代用品人工麝香对H2O2诱导人脐静脉内皮细胞损伤保护作用比较研究

目的：研究麝香（TR）及其代用品人工麝香（RG）对H₂O₂诱导人脐静脉内皮细胞（HUVECs）损伤保护作用及机制。方法：将对数期生长的HUVECs细胞随机分为5组：空白对照、H₂O₂、H₂O₂+NAC、H₂O₂+TR和H₂O₂+RG。HUVECs细胞在H₂O₂作用2 h后,采用MTS法检测细胞增殖光密度值并计算存活率,利用试剂盒测定细胞外液LDH漏出量、细胞内MDA含量、SOD和GSH-Px活力。对HUVECs细胞进行伊红染色,然后用光学显微镜观察细胞形态变化。结果：与H₂O₂组相比较,麝香（50 μg·mL^-1）增加了细胞存活率（P<0.05）;使LDH漏出量和MDA含量显著降低（P<0.05,P<0.001）;使SOD和GSH-Px活力显著提高（P<0.01,P<0.05）;抑制了H₂O₂对HUVECs细胞形态的改变,减少了细胞内ROS水平。人工麝香（50 μg·mL^-1）对HUVECs细胞也有保护作用,但不具有统计学意义。结论：麝香对H₂O₂诱导的人脐静脉内皮细胞损伤具有保护作用,作用机制可能与有效改善细胞内抗氧化酶的活性,减轻氧化应激损伤有关。此外,麝香对人脐静脉内皮细胞的保护作用略强于人工麝香。     

血管紧张素-(1-7)对血管紧张素Ⅱ致人脐静脉内皮细胞损伤的保护作用研究

目的观察血管紧张素-(1-7) [Ang-(1-7)]对血管紧张素II(Ang II)致人脐静脉内皮细胞(HUVECs)损伤的保护作用.方法体外培养的HUVECs随机分为4组对照组,AngII组,Ang-(1-7)组,AngII+Ang-(1-7)组.采用分光光度计测定培养的HUVECs乳酸脱氢酶(LDH)漏出;流式细胞仪检测细胞凋亡;硝酸还原酶法和放射免疫分析技术分别测定HUVECs上清液中一氧化氮(NO)和内皮素-1(ET-1)的含量.结果与对照组比较,Ang II(0.1 μmol·L-1)显著增加HUVECs LDH漏出(P＜0.01)、ET-1分泌(P＜0.01)和HUVECs凋亡率(P＜0.01),显著减少NO的含量(P<0.05);Ang-(1-7)呈剂量依赖性抑制了Ang II的促LDH漏出、ET-1分泌、增加细胞凋亡等作用,同时明显促进HUVECs的 NO释放;单用Ang-(1-7)对HUVECs无明显影响.结论 Ang-(1-7)可抑制Ang II所致的体外培养HUVECs损伤,对内皮细胞具有保护作用.     

左旋龙脑对人脐静脉内皮细胞氯通道和细胞容积的影响

目的研究左旋龙脑对人脐静脉内皮细胞株(human umbilical vein endothelial cells,HUVECs)氯通道和细胞容积的作用。方法膜片钳技术记录全细胞氯电流;siRNA干扰技术下调ClC-3表达;动态图像分析测量细胞容积。结果 20 nmol·L~(-1)左旋龙脑能明显激活HUVECs氯电流,电流密度达(79.59±4.90)pA/pF,左旋龙脑激活的电流能被氯通道阻断剂NPPB、DIDS抑制,外向电流抑制率分别为(95.57±2.57)%、(97.28±6.36)%;下调ClC-3氯通道表达后,左旋龙脑激活的氯电流明显减小至(27.03±3.89)pA/pF;左旋龙脑能明显诱导细胞容积缩小(14.38±1.58)%,氯通道阻断剂NPPB阻断左旋龙脑诱导的细胞容积改变。结论左旋龙脑能激活HUVECs的ClC-3氯通道,使Cl-外流诱发细胞容积减小。     

Generation of complement molecular complex C5b-9 (C5b-9) in response to poly-traumatic hemorrhagic shock and evaluation of C5 cleavage inhibitors in non-human primates

Severe trauma initiates a systemic inflammatory cascade and that involves early activation of complement and cleavage of C5 into C5a (anaphylatoxin) and C5b (C5b-9 membrane attack complex). We examined activation of C5 in non-human primate (NHP) models of hemorrhagic shock.Blood plasma concentrations of C5b-9 were significantly increased in NHPs in response to hemorrhage alone and were further increased with the addition of tissue trauma. The onset of increased C5 cleavage was accelerated in NHPs that experienced decompensated poly-traumatic hemorrhagic shock. Next, to identify an effective inhibitor of NHP C5 cleavage in vitro, as a first step in the development of a potential therapy, three inhibitors of human C5 cleavage and hemolysis were tested in vitro. NHP C5 cleavage and complement-mediated hemolysis were successfully inhibited by pre-treatment of serum samples with a small, inhibitory peptide RA101348. Commercially-available C5 inhibitory antibodies were found to exhibit species-specific efficacy in vitro. Quidel's A217 antibody demonstrated dose-dependent inhibition of C5 cleavage and hemolysis in NHP samples, whereas LGM-Eculizumab only inhibited complement-mediated hemolysis in human samples.This study shows that complement activation in NHPs following experimental poly-traumatic hemorrhagic shock is consistent with clinical reports, and that cleavage of C5 and complement-mediated hemolysis can be effectively inhibited in vitro using a small peptide inhibitor. Taken together, these findings offer a clinically-relevant vehicle and a potential strategy for treatment of hemorrhagic shock with poly-traumatic injury.     

辛伐他汀对脐静脉内皮细胞金属基质蛋白酶9表达的影响

目的观察辛伐他汀对脐静脉内皮细胞（human umbilical vein endothelial cell,HUVEC）金属基质蛋白酶9（matrix metalloproteinase-9,MMP-9）表达的影响。方法采用逆转录聚合酶链反应及蛋白质免疫印迹分析检测MMP-9mRNA转录和蛋白水平表达,观察辛伐他汀不同浓度及不同孵育时间HUVECMMP-9表达的影响。结果辛伐他汀呈浓度和时间依赖性减低HU-VEC的MMP-9mRNA转录和蛋白水平的表达。结论辛伐他汀可抑制HUVE CMMP-9表达,防治动脉粥样硬化。     

人脐静脉内皮细胞水通道蛋白9磷酸化与砷耐受性的关系

目的探讨人脐静脉内皮细胞株ECV-304和抗砷性ECV-304(AsRE)细胞株中水通道蛋白AQP9的表达及其磷酸化水平以及其对亚砷酸钠(NaAsO2)耐受性之间的关系。方法 采用CCK8法测定NaAsO22.5～40μmo.l L-1作用24 h对ECV-304和AsRE细胞株的毒性。NaAsO22.5～20μmol.L-1处理ECV-304和AsRE细胞株2 h,实时定量PCR法测定AQP9 mRNA表达水平,Western印迹法检测AQP9和p38蛋白表达水平;免疫印迹法检测AQP9蛋白磷酸化水平;电感耦合等离子体质谱仪(ICP-MS)法测定细胞内砷含量。结果 NaAsO2在AsRE和ECV-304细胞的IC50分别为12.59和4.06μmol.L-1。在NaAsO2同浓度下,AsRE细胞内砷摄入量均低于ECV-304细胞(P<0.05)。NaAsO2处理ECV-304和AsRE细胞后,AQP9 mRNA表达水平均有所下调,但在AsRE细胞中的下调幅度显著高于ECV-304细胞(P<0.05);AQP9蛋白水平在AsRE细胞呈浓度增加而下降,而ECV-304细胞中则无明显变化;ECV-304细胞中AQP9磷酸化水平随浓度有所上升,AsRE细胞中AQP9磷酸化水平则一直维持在更高的表达水平。NaAsO2处理后,p38蛋白及其磷酸化水平在两个细胞株间无明显变化。结论 AsRE细胞中AQP9的磷酸化水平与砷耐受性有明显相关性,其可能机制是AQP9通过影响细胞对砷的摄入影响砷的耐受性。     

Testing endodontic sealers on human umbilical vein endothelial cells

The toxic potential of the endodontic sealers ingredients, especially the unreacted monomer, that can irritate the periapical tissue and interfere with the healing process, thus having a negative impact on the biocompatibility of the material. The aim of this study was to evaluate the influence of three experimental endodontic sealers on cells viability in vitro. Human umbilical vein endothelial cells (HUVEC) were used. The experiments were done with solid samples and extracts of sealers in artificial saliva and water. The experiments evaluated the cytotoxicity of the residual monomers that resulted from the tested composites. The decrease in cell viability was quantified by colorimetric measurement of formazan. The components of the sealers dissolved in artificial saliva and water were determined by high performance liquid chromatography (HPLC). The HUVEC are a novelty for testing the endodontic sealers biocompatibility, with certain advantages compared to other cell types used in the literature, e.g. HELA cells, fibroblasts. The data showed that cytotoxicity was directly linked with the unreacted monomer — 2-hydroxyethyl methacrylate (HEMA) present in these composites. Two of the three formulations had little or no cytotoxic effect, which makes them suitable for further testing in order to be used in endodontic treatment.     

Apoptosis of human umbilical vein endothelial cells induced by artesunate

Artesunate (ART), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, is an effective novel antimalarial drug. The present study investigated the apoptotic activity of artesunate in cultured human umbilical vein endothelial cell (HUVEC) by means of nuclear staining, DNA agarose gel electrophoresis, and flow cytometry. The observations also indicated that artesunate induced apoptosis of HUVEC in a concentration-dependent and time-dependent manner. A Western immunoblot analysis showed down-regulation of the bcl-2 protein and up-regulation of the bax protein in the artesunate-treated HUVEC. Ca2+ in cells was evaluated by fluorescent spectrophotometer using Fura 2-AM as probe. These results suggest that artesunate may be a potential apoptosis-inducing agent for endothelial cells.     

Desensitization of agonist-stimulated prostacyclin release in human umbilical vein endothelial cells

Prostacyclin (PGI2) release was studied in perfused columns of human umbilical vein endothelial cells cultured on microcarrier beads. Substantial homologous desensitization of PGI2 release occurred when cells were exposed to agonist for 2 min after a previous exposure; the extent depended on the concentration and duration of the first challenge. Recovery from exposure to ATP or bradykinin was complete in less than 80 min; recovery from thrombin was incomplete after greater than 80 min, and this was apparently related to its proteolytic activity. Experiments with ibuprofen, a reversible inhibitor of cyclo-oxygenase, demonstrated that homologous desensitization did not involve inactivation of cyclo-oxygenase. ATP and bradykinin did not induce heterologous desensitization. Thrombin and trypsin induced cross-desensitization, but neither agonist significantly reduced responses to ATP or bradykinin, suggesting that a common proteolytic mechanism is responsible for their ability to induce PGI2 synthesis. We conclude that desensitization of PGI2 release in response to physiological agonists is generally agonist-specific and involves modulation of molecular events at or close to the receptors involved, rather than inactivation of prostanoid biosynthesis.     

青蒿琥酯诱导人脐静脉内皮细胞凋亡

目的　探讨青蒿琥酯对人脐静脉内皮细胞(HUVEC)的诱导凋亡作用 ,以及这种诱导作用与内皮细胞增殖及细胞内游离Ca2 + 的关系。方法　采用MTT比色法测定青蒿琥酯对HUVEC增殖的影响 ,Fura 2 /AM荧光标记法测定HUVEC胞内游离Ca2 + 浓度 ([Ca2 + ]i) ,应用吖啶橙 /溴化乙啶荧光双染色、DNA琼脂糖凝胶电泳、流式细胞术检测细胞凋亡。结果　青蒿琥酯可抑制HUVEC增殖 ,明显诱导HUVEC凋亡 ,并呈浓度依赖性和时间依赖性效应关系。在发生凋亡的同时伴有 [Ca2 + ]i 显著升高。结论　青蒿琥酯可诱导HUVEC凋亡 ,其机制可能与[Ca2 + ]i 升高有关