黄连素对人脐静脉内皮细胞分泌一氧化氮的影响

目的 探讨黄连素对高糖、高脂损伤人脐静脉内皮细胞(HUVECs)分泌一氧化氮的影响及其保护内皮细胞的作用机制.方法 体外培养HUVECs,采用MTT法检测细胞生长存活率,并建立Glu+ ox-LDL损伤HUVECs模型.实验分组如下:正常组:DMEM培养液+10％胎牛血清;模型组:Glu 40 mmol/L+ ox-LDL 100 mg/L;黄连素低组:Glu 40 mmol/L+ ox-LDL 100 mg/L+黄连素1.25 μg/ml;黄连素中组:Glu 40 mmol/L+ ox-LDL 100 mg/L+黄连素2.5μg/ml;黄连素高组:Glu 40 mmol/L+ ox-LDL 100 mg/L+黄连素5μg/ml.采用硝酸还原酶法检测培养HUVECs上清液中NO含量,比较各组细胞NO的分泌水平.结果 模型组NO水平显著低于正常对照组(P＜0.01),黄连素处理组NO水平显著高于模型组,以5 μg/ml剂量组作用最显著(P＜0.01).结论 高糖、高脂处理HUVECs,可以降低HUVECs存活率,减少NO释放;黄连素可以提高Glu、ox-LDL联合诱导的HUVECs损伤的活性,并提高增殖率;黄连素改善Glu、ox-LDL联合诱导的HUVECs损伤的作用机制与其促进NO释放有关.     

硒对人脐静脉内皮细胞ECV-304单核细胞趋化蛋白-1表达的影响

目的 观察硒对人脐静脉内皮细胞ECV-304单核细胞趋化蛋白-1(MCP-1)表达的影响方法分别用高葡萄糖、糖基化终末产物，高胰岛素和过氧化氢孵育人脐静脉内皮细胞ECV-304；在先加入100nmol／L硒后，再给予以上4种因素．分别检测人脐静脉内皮细胞ECV0304MCP-1mRNA的表达并比较。结果 4种因素均可作为独立因素，导致人脐静脉内皮细胞ECV304MCP-1mRNA表达量增加：硒能抑制4种因素所致的MCP-1mRNA表达。结论 硒抑制MCP-1 mRNA在人脐静脉内皮细胞ECV-304中的表达。     

卡维地洛预处理的人脐静脉内皮细胞低密度脂蛋白诱导损伤情况观察

目的 观察卡维地洛预处理的人脐静脉内皮细胞（HUVECs）氧化性低密度脂蛋白（ox-LDL）诱导损伤情况。方法 体外培养HUVECs,将细胞分为5组,A、B、C组分别加入5、10、20μmol/L卡维地洛预处理6 h,再加入100μg/mL的ox-LDL诱导培养24 h;D组仅加入100μg/mL的ox-LDL诱导培养24 h;E组加入不含细胞的DMEM（高糖）培养基。采用微板法检测各组细胞LDH活性,CCK-8法测算细胞增殖抑制率,油红O染色观察细胞内脂滴形成情况。结果 与E组比较,B、C、D组LDH活性增强（P均<0.05）;与D组比较,A、B、C组LDH活性减弱（P均<0.05）。与E组比较,C、D组OD值减小（P均<0.05）;与D组比较,A、B、C组细胞OD值及增殖率增加（P均<0.05）;与A组比较,B细胞OD值及增殖率减小（P均<0.05）。与E组比较,D组细胞内红色脂滴积累显著增加;与D组比较,A、B、C组细胞胞内脂滴形成明显减轻,其中以A组抑制脂滴积累效果最为明显。结论 卡维地洛预处理可以减轻ox-LDL诱导HUVECs损伤,尤以浓度为5...     

黄芩苷对ox-LDL诱导的人脐静脉内皮细胞损伤的保护作用

目的观察黄芩苷对氧化型低密度脂蛋白(ox-LDL)诱导的血管内皮细胞的保护作用。方法将体外培养的人脐静脉内皮细胞(EAhy926)分成对照组、ox-LDL组、黄芩苷组和不同浓度的黄芩苷(25、50和100 mg/L)+ox-LDL组,培养24 h。采用CCK-8检测细胞活力,ELISA检测细胞培养上清液中TNF-α和IL-6水平,Western blot检测凋亡相关蛋白Bax和Bcl-2的表达。结果与ox-LDL组相比,50、100 mg/L黄芩苷+ox-LDL组细胞存活率明显升高(P0.05),细胞培养上清中TNF-α和IL-6水平均明显下降(P0.05),Bax/Bcl-2比值降低(P0.05)。结论黄芩苷对预防内皮细胞损伤的保护功能可能是通过抑制炎症因子,减少细胞凋亡,增强细胞活性实现的。     

P38信号通路对人脐静脉内皮细胞单核细胞趋化蛋白-1表达的影响

目的 探讨P38信号通路(P38MAPK)和单核细胞趋化蛋白-1(MCP-1)的关系，以及P38MAPK在糖尿病(DM)动脉粥样硬化(AS)中的作用。方法 分别以高葡萄糖(HG)、糖基化终产物(AGEs)、高胰岛素(HIns)和过氧化氢(H2O2)孵育人脐静脉内皮细胞(HUVECs)，观察HUVECs P38MAPK的蛋白表达和MCP-1 mRNA表达；以P38MAPK特异抑制剂SB203580预处理，再用以上4种刺激因素孵育HUVECs，观察MCP-1 mRNA在HUVECs的表达。结果 HG、AGEs、HIns和H2O2均可独立激活P38MAPK，使磷酸化P38MAPK蛋白表达量及MCP-1 mRNA表达量增加；SB203580预处理后，MCP-1 mRNA表达被明显抑制。结论 P38MAPK调控MCP-1的表达，表明P38MAPK可能是DM致AS发生的始动信号之一。     

氟伐他汀和辛伐他汀对人脐静脉内皮细胞黏附分子-1表达的影响

目的观察氟伐他汀和辛伐他汀对肿瘤坏死因子(TNFα)诱导的人脐静脉内皮细胞(HUVEC)血管细胞黏附分子-1(VCAM-1)表达的影响,以期探讨3-羟-3-甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂可能的非调脂抗动脉粥样硬化作用。方法体外培养HUVEC,加TNFα100U及1×10     

高糖对人脐静脉内皮细胞表面血栓调节蛋白表达及活性的影响

目的观察高糖对血栓调节蛋白在原代人脐静脉内皮细胞的表达和活性的影响。方法体外培养人脐静脉内皮细胞,实验分为三组:1对照组;210 mmol/L葡萄糖组;320 mmol/L葡萄糖组,孵育细胞24 h。分别用流式细胞术、实时荧光定量聚合酶链反应和酶标仪检测血栓调节蛋白的蛋白、m RNA表达及活性强度。结果20 mmol/L葡萄糖组和10 mmol/L葡萄糖组相比于对照组在血栓调节蛋白的蛋白(41.38±3.41、32.60±2.59比27.96±1.58)、m RNA(2.05±0.19、1.44±0.32比1)水平上均升高(P0.05),20 mmol/L葡萄糖组与对照组相比在血栓调节蛋白活性上也增强(0.4157±0.0129比0.3957±0.0100,P0.05),但10 mmol/L葡萄糖组与对照组在血栓调节蛋白活性上差异无显著性。结论高糖可增加血栓调节蛋白表达,并增强其活性,提示这可能为内皮细胞对于高糖的一种防御机制。     

氨氯地平对人脐静脉内皮细胞单核细胞趋化蛋白1表达的影响

目的为研究氨氯地平独立于降压外的血管保护作用,观察其对人脐静脉内皮细胞单核细胞趋化蛋白1表达的影响,进一步明确其抗动脉粥样硬化作用的可能机制。方法用不同浓度氨氯地平预处理人脐静脉内皮细胞1h,与氧化型低密度脂蛋白共同孵育24h后,采用逆转录聚合酶链反应检测氨氯地平对体外培养的人脐静脉内皮细胞单核细胞趋化蛋白1mRNA表达的影响,酶联免疫试剂盒检测培养基中单核细胞趋化蛋白1活性。结果氧化型低密度脂蛋白上调人脐静脉内皮细胞单核细胞趋化蛋白1的表达,各浓度氨氯地平处理组人脐静脉内皮细胞单核细胞趋化蛋白1mRNA与蛋白表达均明显降低(P<0.05)。结论氨氯地平呈浓度依赖性抑制氧化型低密度脂蛋白刺激导致的人脐静脉内皮细胞单核细胞趋化蛋白1表达上调。     

组织因子对人脐静脉内皮细胞单核细胞趋化蛋白-1表达的影响

目的通过观察不同浓度组织因子(TF)对人脐静脉内皮细胞单核细胞趋化蛋白-1(MCP-1)表达的影响,探讨TF对MCP-1的作用及可能机制。方法体外培养人脐静脉内皮细胞,分为对照组及10、100、103、104 ng/L的TF处理组,对照组加入生理盐水,4个处理组分别加入相应浓度的组织因子,分别采用ELLSA法和RT-PCR法检测人脐静脉内皮细胞MCP-1和MCP-1 mRNA的表达。结果 ELLSA和RT-PCR的检测结果显示各组人脐静脉内皮细胞均有MCP-1、MCP-1 mRNA表达,MCP-1和MCP-1 mRNA的表达水平为对照组<10 ng/L组<100 ng/L组<103 ng/L组<104 ng/L组(均P<0.05)。结论 TF呈浓度依赖性的促进人脐静脉内皮细胞MCP-1的表达。     

抵抗素对原代培养的脐静脉内皮细胞分泌ET-1、TGF-β1的影响

目的观察抵抗素对原代培养的脐静脉内皮细胞（HUVECs）产生内皮素-1（ET-1）和转化生长后子-β1（TGF-β1）的影响。方法分别用含不同浓度抵抗素、抗抵抗素IgG加高浓度抵抗素、脂联素加高浓度抵抗番的无血清RPMI 1640液培养HUVECs 48h,用放射免疫法测定其上清液中的ET-1水平,用ELISA法测定爿TGF-β1水平。结果抵抗素呈剂量依赖性促进HUVECs分泌ET-1（P〈0.01）和TGF-β1（P〈0．05）。抗抵抗京IgG能够完全抑制高浓度抵抗索引起的ET-1和TGF-β1分泌（P〉0．05）。生理剂量的脂联素对高浓度抵抗素引起的ET-1的分泌无明显抑制作用（P〉0．05）,对高浓度抵抗素引起的TGF-β1：分泌有部分抑制作用（P〈0．05）。结论抵抗素导致血管内皮细胞功能紊乱。促发糖尿病血管病变。而脂联素在糖尿病血管病变中起保护作用。     

miR-320通过下调SRY相关高迁移率族蛋白4的表达抑制人脐静脉内皮细胞内皮间质转化

目的 探讨miR-320对人脐静脉内皮细胞(HUVEC)内皮间质转化(EndMT)的影响及其调控机制.方法 用miR-320 mimics或miR-320 inhibitor处理HUVEC.Western blot检测内皮细胞标记物血小板内皮细胞黏附分子1(CD31)、血管内皮钙黏蛋白(VE-Cadherin)及间质细...     

罗格列酮对高胰岛素培养的内皮细胞一氧化氮的影响及其机制研究

目的观察罗格列酮（RGZ）对高胰岛素培养的人脐静脉内皮细胞（HUVEC）NO浓度和内皮型一氧化氮合酶（eNOS）、磷酯酰肌醇3激酶（P13K）和蛋白激酶B（PKB）表达的影响,探讨RGZ改善高胰岛素状态下内皮功能障碍的信号转导机制。方法高浓度胰岛素培养HUVEC72h,并用不同浓度的RGZ进行干预。检测NO浓度,PI3K mRNA的表达,PKB、eNOS总蛋白和PKB丝氨酸473（PKB-Ser473）、eNOS丝氨酸1177（eNOS-Ser1177）的磷酸化表达。结果高浓度胰岛素培养HUVEC能呈剂帚和时间依赖性地降低N0的浓度,抑制内皮细胞P13KmRNA表达和PKB-Ser473、eNOS-Ser1177的磷酸化。用RGZ干预能硅著升高高胰岛素培养的内皮细胞NO的浓度和PKB、eNOS的磷酸化,增强PI3KmRNA表达;eNOS和P13K阻断剂均能阻断RGZ对高胰岛素培养的内皮细胞中NO浓度的升高,PI3K阻断剂还能阻断RGZ对高胰岛素培养内皮细胞PKB、eNOS的磷酸化。结论高胰岛素能下调P13K／PKB／eNOs信号通路而抑制内皮细胞NO的产生,RGZ能通过上调PI3K／PKB通路而增强高胰岛素培养的内皮细胞eNOS的活性和NO的产生。     

罗格列酮对内皮细胞单核细胞趋化蛋白-1和血管细胞黏附分子-1表达的影响

目的观察罗格列酮(RGZ)对高糖及C反应蛋白(CRP)诱导的人脐静脉内皮细胞(HUVECs)的单核细胞趋化蛋白-1(MCP-1)及血管细胞黏附分子-1(VCAM-1)mRNA和蛋白表达的影响。方法体外培养HUVECs,细胞传至5代,随机分为7组,正常对照组(C组),高糖组(HG组),高糖+CRP组(HGC组),CRP组,高糖+RGZ组(HGR组),高糖+CRP+RGZ组(HGCR组),CRP+RGZ组(CRPR组)。采用RGZ 5.0μmol/L干预HUVECs24 h,RT-PCR、Western blot法分别检测干预前后MCP-1、VCAM-1 mRNA和蛋白的表达水平。结果HG组、HGC组、CRP组HUVECs中MCP-1、VCAM-1的mRNA和蛋白水平较C组显著升高(P<0.01);RGZ干预后,HGR组、HGCR组、CRPR组分别较HG组、HGC组、CRP组MCP-1、VCAM-1的mRNA和蛋白水平显著降低(P<0.01)。结论RGZ通过降低HUVECs中MCP-1、VCAM-1的表达,延缓糖尿病动脉粥样硬化的进程。     

Activated protein C inhibits high mobility group box 1 signaling in endothelial cells

A pathogenic role for high-mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Activated protein C (APC) is the only drug approved by the Food and Drug Administration for severe sepsis; however, its effect on HMGB1 signaling has never been investigated. Here, we monitored the effect of APC on the lipopolysaccharide-mediated release of HMGB1 and the HMGB1-mediated modulation of proinflammatory responses in HUVECs. APC potently inhibited the release of HMGB1 and down-regulated the adhesion of the monocytic cell line, THP-1, to HMGB1-activated endothelial cells. HMGB1 up-regulated proinflammatory responses by interacting with 3 pathogen-related pattern recognition receptors: TLR2 and TLR4 and the receptor for advanced glycation end products. APC not only inhibited HMGB1 release but also down-regulated the cell surface expression of all 3 HMGB1 receptors in endothelial cells. The protective effects of APC were mediated through endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR-1). Interestingly, a thrombin derivative containing the Gla-domain of APC recapitulated all protective effects of APC with a 20- to 50-fold higher efficacy. These results suggest that the EPCR- and PAR-1-dependent protective effects of APC in severe sepsis may partially be mediated through the inhibition of HMGB1 signaling and that the chimeric thrombin mutant has potential therapeutic utility for severe sepsis.     

辛伐他汀对内皮细胞PD-L1表达的影响

目的研究辛伐他汀对受氧化低密度脂蛋白（ox-LDL）干预的人脐静脉内皮细胞程序性死亡配体1（PD-L1）表达的影响。方法体外培养人脐静脉内皮细胞,用50 mg/L ox-LDL含或不含5μmol/L辛伐他汀共同孵育温育人脐静脉内皮细胞12 h,RT-PCR及流式细胞术分别检测人脐静脉内皮细胞PD-L1 mRNA及蛋白表达水平。结果与对照组比较,ox-LDL刺激人脐静脉内皮细胞PD-L1表达明显升高（P〈0.01）;预先给予辛伐他汀可明显抑制ox-LDL诱导的PD-L1表达,与单纯ox-LDL组比较具有统计学差异（P〈0.05）。结论PD-L1参与了辛伐他汀的免疫抑制作用。     

瘦素对人脐静脉内皮细胞血管内皮生长因子表达的影响

目的:探讨瘦素对人脐静脉内皮细胞(HUVECs)血管内皮生长因子(VEGF)表达的影响。方法:用不同浓度的瘦素刺激原代培养的HUVECs,检测HUVECs表达VEGF的情况。结果:在相同作用时间下,随着瘦素浓度的升高,VEGF蛋白及VEGF mRNA的表达也随之升高,经统计学检验有相关性;在相同瘦素浓度下, 随着作用时间的延长,VEGF蛋白及VEGFmRNA的表达也随之升高,且有相关性。结论:瘦素可以刺激HU- VECs表达VEGF而且呈时间和剂量相关性。     

Effects of bone morphogenetic protein 2 on human umbilical vein endothelial cells

Bone morphogenetic proteins (BMPs) and their receptors play important roles in cellular processes such as proliferation, differentiation, migration and cell survival. It was also demonstrated that BMPs are involved in vasculogenesis and angiogenesis. In this study, we investigated the expression profile of BMP receptors in human umbilical vein endothelial cells (HUVECs) and determined the effect of BMP-2 on proliferation, migration, invasion, cell survival and tube formation. HUVECs express the type I BMP receptors ALK2, ALK3 and ALK6 and the type II receptor BMPR-II. Treatment of HUVECs with recombinant human BMP-2 induced migration, invasion and tube formation of HUVECs without affecting proliferation and apoptosis. Our data suggest that BMP-2 represents a chemoattractant and proangiogenic factor for HUVECs.     

Influence of intercellular junctions on endothelin secretion of human umbilical vein endothelial cells in vitro

The endothelium plays a pivotal role in the theological regulation of blood flow by the secretion of vasoactive factors. The interaction between shear forces and the endothelium is determined by the mechanical properties of the endothelial cell layer which are associated with intercellular junctions. Cell-cell contacts could therefore modulate the secretion of vasocative factors in response to theological stimuli. We investigated the relationship between intercellular junctions and the secretion of the vasoconstrictor peptide endothelin and the coagulation co-factor von Willebrand factor (vWF). Human umbilical vein endothelial cells (HUVECs) were used as in vitro endothelial model system. Intercellular junctions were reversibly disrupted by calcium chelation or hypertonic stress; alternatively, the formation of intercellular junctions was inhibited by culturing the cells in suspension or by plating them in the presence of an inhibitory anti-VE-cadherin antibody. The opening of intercellular junctions was verified by assessing transmonolayer electrical resistance (TMR) and immunofluorescence morphology. The concentration of endothelin and vWF was measured in the cell culture supernatants using specific ELISAs. The secretion of endothelin was inhibited by EGTA (5 mM) and stimulated by incubation with tumor necrosis factor α (TNFα, 40 ng/ml). Treatment with hypertonic medium (glycerol, 1200 mosmol/l) for 10 minutes opened intercellular junctions and markedly reduced the secretion of endothelin. HUVECs in suspension culture did not secrete endothelin and failed to respond to TNFα, but readily resumed these functions upon forming a new monolayer on plastic. The reconstitution of intercellular junctions after suspension culture could be inhibited using a specific anti-VE-cadherin antibody. This antibody, but not a non-specific anti-humanIgG antibody reduced endothelin secretion. The secretion of von Willebrand Factor was less dependent on intercellular junctions. The opening of intercellular junctions did not induce cell death, since the cells continued to exclude trypan blue. The results of this study suggest a novel and potentially pathophysiologically/clinically relevant correlation between intercellular junctions and the secretion of endothelin in endothelial cells. Received: 17 December 1999, Returned for revision: 20 January 2000, Revision received: 14 February 2000, Accepted: 2 March 2000