西洛他唑对人脐静脉内皮细胞血管细胞黏附分子1和细胞间黏附分子1mRNA表达的影响

目的观察西洛他唑对人脐静脉内皮细胞（HUVECs）血管细胞黏附分子1（VCAM-1）和细胞间黏附分子1（ICAM-1）mRNA表达的影响,探讨西洛他唑可能的抗动脉粥样硬化作用机制。方法将HUVECs用不同浓度的西洛他唑（0μg/L、0.05μg/L、0.1μg/L、1.0μg/L、10μg/L）溶液处理1小时后,用肿瘤坏死因子α（TNF-α）10μg/L诱导24小时。半定量复合逆转录聚合酶链反应（RT-PCR）测定黏附分子VCAM-1和ICAM-1mRNA的表达。结果 TNF-α能上调VCAM-1和ICAM-1的表达,西洛他唑在一定程度上可抑制上述作用,随着西洛他唑浓度的增加,ICAM-1mRNA表达水平逐步下降,分别为0.239±0.012、0.205±0.012、0.166±0.010、0.136±0.008,VCAM-1mRNA表达水平也逐步下降,分别为0.114±0.048、0.093±0.051、0.083±0.045、0.068±0.039。结论西洛他唑可抑制TNF-α诱导的HUVECs的黏附分子VCAM-1和ICAM-1mRNA表达,提示西洛他唑的抗动脉粥样硬化作用可能是通过阻止血单核细胞向血管内皮细胞聚集和黏附实现的。     

过表达miR-338-3p对炎症信号通路的影响

目的研究过表达miR-338-3p在内皮细胞系EOMA中对炎症信号通路的影响。方法将EOMA细胞分为四组即对照组、miR-338-3p过表达组、TNF-α处理组、miR-338-3p与TNF-α共处理组。用Real time PCR检测miR-338-3p及黏附分子VCAM-1和ICAM-1mRNA表达水平,Western blot分析ERK/p38 MAPK信号通路活性。结果与对照组比较,miR-338-3p过表达组的黏附分子VCAM-1及ICAM-1mRNA表达水平和ERK/p38信号通路活性明显降低,而TNF-α处理组的黏附分子VCAM-1及ICAM-1mRNA表达水平和ERK/p38信号通路活性明显升高;miR-338-3p与TNF-α共处理组与TNF-α处理组比较,黏附分子VCAM-1及ICAM-1mRNA表达水平和ERK/p38信号通路活性明显降低;miR-338-3p与TNF-α共处理组与miR-338-3p过表达组比较,黏附分子表达水平及ERK/p38信号通路活性不再升高。结论过表达miR-338-3p抑制ERK/p38信号通路活性,降低黏附分子VCAM-1和ICAM-1mRNA表达水平;过表达miR-338-3p能够逆转TNF-α对ERK/p38通路的激活和TNF-α对黏附分子VCAM-1及ICAM-1mRNA表达的促进作用。     

蛋白激酶C对血管内皮功能的影响

【目的】研究血管内皮蛋白激酶C对内皮通透性及粘附功能的影响。【方法】以血管内皮细胞为研究对象,以流式分析法测定细胞间黏附分子-1（ICAM-1）,血管细胞黏附分子-1（VCAM-1）的表达;以同位素标记法测定HUVECs的粘附率;双室培养法测定HUVECs的通透率。【结果】10ng／mlPMA处理HUVECs后,HUVECs的通透性于30min时开始明显增高;VCAM-1,ICAM-1表达增强,对血小板粘附率2h达到最大值。【结论】PKC活化能其上调血管内皮细胞VCAM-1、ICAM-1的表达,并增强其粘附性。HUVECs的PKC活化与血管内皮细胞通透性增加有关。     

阿托伐他汀钙调节人脐静脉内皮细胞E-选择素和细胞间黏附分子1表达的浓度依赖性

目的：观察具有调脂作用的阿托伐他汀钙在不同浓度下对肿瘤坏死因子诱导体外培养的人脐静脉内皮细胞表达E-选择素和细胞间黏附分子1的影响。 方法：①实验于2002-05／2003-12在大连医科大学附属第二医院实验室完成。将健康婴儿脐带进行无菌处理，用D-Hank’s液冲洗脐静脉，1g/L胶原酶消化、离心，置于37℃、体积分数0．05 CO2培养箱中培养，选择第2,3代细胞作为实验用。②用肿瘤坏死因子40μg／L、不同浓度（0．005～10μmol／L）的阿托伐他汀钙与体外培养的人脐静脉内皮细胞共同孵育24h后，提取细胞的总RNA，采用半定量反转录聚合酶链反应在mRNA水平进行分析。③用曲线图表示E-选择素和细胞间黏附分子1表达在不同浓度阿托伐他汀钙影响下的变化趋势。 结果：①在mRNA水平，阿托伐他汀钙对肿瘤坏死因子诱导的人脐静脉内皮细胞所表达的E-选择素与细胞黏附分子1均具有浓度依赖性。②阿托伐他汀钙对肿瘤坏死因子诱导人脐静脉内皮细胞表达E-选择素mRNA呈促进作用，即阿托伐他汀钙在0～10μmol／L的浓度区间内，随着其浓度由0，0．005,0．01μmol／L渐升至10μmol／L，E-选择素mRNA的表达呈逐渐升高的趋势。③阿托伐他汀钙对肿瘤坏死因子诱导人脐静脉内皮细胞表达细胞间黏附分子1的影响呈双向性，具有浓度差异性。即随着阿托伐他汀钙浓度由0,0．005,0．01μmol／L升至0．05μmol／L，细胞间黏附分子1 mRNA表达呈逐渐下降趋势，而在高浓度（0．05-10μmol／L）区间，随着阿托伐他汀钙浓度0．05，0．1μmol／L渐升至10μmol／L，细胞间黏附分子1 mRNA的量呈逐渐升高的趋势。 结论：在基因水平，阿托伐他汀钙对肿瘤坏死因子诱导血管内皮细胞黏附分子的表达有着确切的调节作用，且存在着浓度依赖性，并在不同的浓度区间，对不同的黏附分子作用不同。     

内吗啡肽-1对正常人骨髓基质细胞造血调控的研究

本研究探讨内吗啡肽-1对正常人骨髓基质细胞表面黏附分子细胞间黏附分子（ICAM-1）和血管细胞黏附分子（VCAM-1）,以及基质细胞分泌的细胞因子IL-6、TNF-α的调控作用。应用流式细胞术检测黏附分子ICAM-1和VCAM-1的表达,用ELISA法检测细胞因子IL-6、TNF-α的浓度。结果表明：加入内吗啡肽-1培养24小时后ICAM-1和VCAM-1表达与对照组比较有显著性差异（p〈0.05）,但内吗啡肽-1浓度的改变对于二者的表达无影响;IL-6、TNF-α的浓度实验各组数据与对照组比较无显著性差异（p〉0.05）。结论：内吗啡肽-1能调控正常骨髓基质表面ICAM-1和VCAM-1的表达;对于正常骨髓基质细胞分泌的细胞因子IL-6、TNF-α无促进或抑制作用。     

雌激素受体α与NF-kappa BP65在Buerger病病变血管内皮细胞的表达和意义

目的：研究血栓闭塞性脉管炎（Buerger病）病变血管内皮细胞中雌激素受体α（ERα）与核转录核因子（NF—kappa B）表达的情况和意义。方法：采用免疫组织化学染色S—P法检测30例男性Buerger病患者动脉血管和30例正常男性动脉血管内皮细胞的ERα、NF—kappa BP65蛋白，细胞间黏附分子-1（ICAM-1）和血管内皮细胞黏附分子-1（VCAM-1）的表达水平。并采用直线回归方法分析其相关性。结果：（1）与正常组相比较，Buerger病患者病变血管内皮细胞ERα、NF—kappa BP65、ICAM1以及VCAM—1表达水平均呈显著性增强（P〈0．05）。（2）60例动脉血管内皮细胞检测数据提示ERα与NF—kappaB表达呈直线回归关系（B=0．706，P〈0．05）；NF-kappa BP65表达与ICAM-1表达呈直线回归关系（b=0．392，P〈0．05）；NF—kappa BP65表达与VCAM—1表达也呈直线回归关系（b=1．094，P〈0．05）。结论：NF—kappaB的激活导致Buerger病患者体内ICAM—1和VCAM-1表达增强，是Buerger病患者血管损伤的起始因素之一。Buerger病患者体内激活的NF—kappaB诱导体内雌激素代偿性增高，进一步促使ERα表达增强，并通过雌激素-ERα途径，抑制NF—kappaB的活性。     

血清黏附分子和细胞因子与2型糖尿病患者血管病变的关系

目的 探讨2型糖尿病（T2DM）患者血清黏附分子与细胞因子水平以及与血管病变的关系.方法 选择96例T2DM患者按照血管病变情况分为无血管病变组（28例）、微血管病变组（33例）、大血管病变组（35例）,选择同期健康体检者30名作为对照组,ELISA法检测血清细胞间黏附分子1（ICAM-1）和血管细胞黏附分子1（VCAM-1）,细胞因子包括白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）和假性血友病因子（vWF）浓度,同时检测治疗半个月后T2DM患者黏附分子和细胞因子的表达.结果 与对照组比较,T2DM患者不同血管病变组ICAM-1、VCAM-1、IL-6和TNF-α水平都明显增高,差异有统计学意义（P均＜0.05）,且随着患者血管病变的严重,其表达也有升高趋势,差异有统计学意义（P均＜0.05）;治疗半个月后,患者血清ICAM-1、VCAM-1、IL-6和TNF-α水平显著性降低,差异具有统计学意义（t值分别为16.281、8.712、7.697、8.445,P均＜0.05）,各指标分别从治疗前（505.34±56.42） μg/L,（570.85±59.54）μg/L,（94.51±18.04） ng/L,（70.57±18.34） ng/L降到治疗后（390.53±45.23） μg/L,（482.93±69.85） μg/L,（77.31±15.49） ng/L,（50.45±12.66） ng/L;T2DM患者血清ICAM-1、VCAM-1、IL-6及TNF-α水平与vWF的表达都呈明显的正相关（r值分别为0.482、0.453、0.576、0.534,P均＜0.05）.结论 黏附分子和细胞因子共同参与了T2DM恶化以及血管病变的发生发展.     

地氟醚预处理对缺氧/复氧损伤时核因子-κB转导通路效应分子的影响

目的：探讨地氟醚预处理对于核因子（NF）-κB信号转导通路激活后效应分子的影响。方法：选用人脐静脉内皮细胞株（ECV304）。将细胞分为5组：（1）空白对照组;（2）缺氧/复氧（A/R）组;（3）A/R＋肿瘤坏死因子-α（TNF-α,10ng.mL-1）刺激组（A/R＋TNF-α组）;（4）地氟醚1.0MAC预处理＋A/R组（Des＋A/R组）;（5）地氟醚1.0MAC预处理＋A/R＋TNF-α（10ng.mL-1）刺激组（Des＋A/R＋TNF-α组）。应用逆转录-聚合酶链反应（RT-PCR）法分别检测各组细胞的细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）和白细胞介素-8（IL-8）的基因表达水平。结果：A/R组较对照组细胞的ICAM-1、VCAM-1和IL-8mRNA表达水平均有所增加;而经地氟醚预处理后,Des＋A/R组细胞的mRNA表达较A/R组有明显降低。结论：地氟醚预处理可抑制TNF-α刺激的内皮细胞表面黏附分子及炎性介质的基因表达水平。     

油茶皂苷C对脐静脉内皮细胞缺氧及复氧损伤时的影响

目的：研究脐静脉内皮细胞(HUVEC)缺氧／复氧损伤(A／R)时，单核细胞与内皮细胞黏附及细胞间黏附分子-1(ICAM-1)mRNA表达并观察油茶皂苷C(SQS-C)对此的影响。方法：内皮细胞A／R前3h，分别加入终浓度为0．1，1．0和10．0μmol／L SQS-C，再A／R 5h，比色法测定内皮细胞与单核细胞的黏附率，RT-PCR法检测内皮细胞ICAM-1 mRNA的表达。结果：A／R时，单核细胞与内皮细胞的黏附显著增加(P&;lt;0．01)：SQS-C能明显降低细胞黏附率，减少内皮细胞ICAM-1 mRNA的表达(P&;lt;0．05)，并呈剂量依赖性。结论：SQS-C能抑制缺氧／复氧损伤的内皮细胞与单核细胞的黏附，减少内皮细胞ICAM-1 mRNA的表达。     

姜黄素脂质纳米粒对人脐静脉内皮细胞细胞因子表达的影响

目的通过检测姜黄素脂质纳米粒对人脐静脉内皮细胞（HUVEC）分泌一氧化氮（NO）与细胞间粘附分子（ICAM-1）表达水平的影响,探讨新剂型对于动脉粥样硬化疾病的抑制作用。方法制备姜黄素脂质纳米粒,培养HUVEC细胞,随机分正常组、模型组、脂质纳米粒空载药组,三组剂量呈梯度的姜黄素脂质纳米粒药物组。10μg／LTNF—α刺激后,加入5-60mg／L姜黄素纳米制剂,用MTT增殖实验检测姜黄素脂质纳米粒对HUVEC细胞增殖作用的影响;采用实时荧光定量PCR测定一氧化氮合酶（eNOS）与ICAM—1的基因表达水平;通过ELISA法观察ICAM-1的含量;用硝酸还原酶法检测No表达量。结果MTT法检测显示5~30mg／L姜黄素脂质纳米粒对细胞活力无明显影响,当药物浓度为60mg／L时,活细胞数量有所减少。与正常组比较,模型组eNOS表达量降低（P〈0．01）,姜黄素脂质纳米粒预处理的HUVEC细胞可显著上调eNOS基因表达（P〈0．01）,促进N0分泌量增多（P〈O．01）;TNF-α引起的血管细胞粘附分子ICAM-1中mRNA的表达上升（P〈0．01）;姜黄素脂质纳米粒预处理的HUVEC细胞ICAM-1mRNA表达量与血清中的ICAM-1蛋白含量比较较模型组有明显下降（P〈0．01）。结论姜黄素脂质纳米粒剂型制备成功,结果显示该剂型可以上调内皮细胞eNOS的表达、促进No的分泌、抑制TNF-α引起的子ICAM-1表达上升,从而发挥内皮细胞保护作用以及抗动脉粥样硬化作用。     

Induction of tissue factor production but not the upregulation of adhesion molecule expression by ceramide in human vascular endothelial cells

Binding of tumor necrosis factor-alpha (TNF-alpha) to p60 TNF-alpha receptor induces the activation of sphingomyelinase to generate ceramide, which in turn activates certain protein kinases and phosphatases, resulting in various TNF-alpha-mediated biological effects. We have investigated the role for the sphingomyelin/ceramide pathway in the TNF-alpha-induced upregulation of adhesion molecule expression and tissue factor production of human endothelial cells. TNF-alpha stimulated human umbilical vascular endothelial cells (HUVECs) to upregulate the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and HLA class I molecules in addition to the induction of procoagulant tissue factor production. C2-ceramide, a highly cell-permeable ceramide analog, was able to stimulate HUVECs to produce tissue factor activity as well as TNF-alpha. However, C2-ceramide did not stimulate HUVECs to upregulate the expression of VCAM-1, ICAM-1 and HLA class I molecules. These results suggest that there exist both the ceramide-dependent and -independent pathways in TNF-alpha signal transduction system in human vascular endothelial cells.     

Effect of gold sodium thiomalate and its thiomalate component on the in vitro expression of endothelial cell adhesion molecules.

Endothelial adhesion molecules play an important role in the tissue recruitment of leukocytes in inflammatory conditions such as rheumatoid arthritis. We have investigated the effect of the antirheumatic drug gold sodium thiomalate on adhesion molecule protein and mRNA expression in cultured human endothelial cells. Gold sodium thiomalate inhibited cytokine (TNF, IL-1, IL-4)-stimulated expression of vascular cell adhesion molecule-1 and E-selectin but not intercellular adhesion molecule-1 on endothelial cells. Gold sodium thiomalate also suppressed TNF-stimulated increases in vascular cell adhesion molecule-1 and E-selectin mRNA levels but had no effect on intercellular adhesion molecule-1 mRNA. Thiomalate (mercaptosuccinate), but not gold thioglucose or D-penicillamine, mimics the effect of gold sodium thiomalate at equimolar concentrations. We propose that the inhibition of vascular cell adhesion molecule-1 and E-selectin expression by gold sodium thiomalate is due to its thiomalate and not its gold component. Gold sodium thiomalate has a direct effect on endothelial adhesion molecule expression, and this may contribute to its antiinflammatory activity.     

Bay w 9798, a dihydropyridine structurally related to nifedipine with no calcium channel-blocking properties, inhibits tumour necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in endothelial cells by suppressing reactive oxygen species generation

Dihydropyridine-based calcium antagonists (DHPs) are widely used to treat hypertension. We have previously shown that nifedipine, one of the most popular DHPs, blocks tumour necrosis factor-alpha (TNF-alpha)-induced monocyte chemoattractant protein-1 as well as vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells by suppressing reactive oxygen species generation (ROS). The molecular mechanism is still to be elucidated, however, because endothelial cells do not possess voltage-operated L-type calcium channels. The aim of this study was to determine in TNF-alpha-exposed human umbilical vein endothelial cells (HUVECs) whether and how Bay w 9798, a dihydropyridine structurally related to nifedipine with no calcium antagonistic properties, may suppress VCAM-1 expression, a key molecule which mediates the adhesion of monocytes to vasculature in the early stages of atherosclerosis. In HUVECs, 10 ng/ml TNF-alpha for 4 h stimulated ROS generation and subsequently upregulated VCAM-1 mRNA levels, both of which were dose-dependently blocked by Bay w 9798. The results demonstrated that Bay w 9798 inhibited VCAM-1 expression in TNF-alpha-exposed cells by suppressing ROS generation. They suggest that the anti-inflammatory and anti-oxidative properties of nifedipine and Bay w 9798 may be ascribed to the dihydropyridine structure, which is common to both molecules and has no calcium antagonistic ability.     

组织因子对人脐静脉血管内皮细胞黏附功能的影响

目的:观察组织因子对体外培养的人脐静脉血管内皮细胞黏附功能的影响。方法:体外培养人脐静脉血管内皮细胞,分别加入不同浓度的组织因子,采用细胞双抗体夹心酶联免疫分析法及ELISA检测血管内皮细胞黏附因子-1(VCAM-1)及可溶性细胞间黏附因子-1(sICAM-1)表达的变化,并用单核细胞黏附率试验检测单核细胞黏附。结果:TF在0～1000pmol/L范围内以剂量依赖方式增强血管内皮细胞VCAM-1和sICAM-1的表达(P<0.01);细胞黏附试验表明TF增加单核细胞黏附率,且黏附率与加入的TF浓度呈正相关,在1000pmol/L时刺激作用最强(P<0.01)。结论:体外情况下TF能上调内皮细胞黏附分子VCAM-1及sICAM-1表达,促进单核细胞黏附,这可能在TF致动脉粥样硬化的病理过程中发挥重要作用。     

Tumor necrosis factor-alpha stimulates attachment of small cell lung carcinoma to endothelial cells

Tumor cell attachment to endothelial cells (ECs) is an important step in the metastasis of small cell lung carcinoma (SCLC). Tumor necrosis factor-alpha (TNF-alpha) stimulation of ECs increases the attachment of some malignant cell types to ECs by affecting the expression of cell adhesion molecules (CAMs). Similarly, the inhibition of EC protein kinase C (PKC) and tyrosine kinase (TK) pathways modulates TNF-alpha-mediated effects on CAM expression. We hypothesized that TNF-alpha would increase SCLC attachment to ECs by affecting CAM expression through activation of PKC and TK pathways. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-alpha (0 to 500 U/mL) for variable time periods (1 to 24 hours), and the attachment of H82 cells (an SCLC cell line) to the HUVECs was quantified. TNF-alpha stimulation of the HUVECs increased H82 attachment from 28.1% +/- 1.6% to 48.8% +/- 1.7% (P < .05). Preincubation of HUVECs with the PKC inhibitors bis-indolylmaleimide (BIN) or calphostin C or the TK inhibitors genistein or herbimycin A (HMA) blocked the TNF-alpha-induced increase in H82 cell attachment. The addition of antibodies to vitronectin (Vn) or beta1-integrin to TNF-alpha-activated HUVECs before the addition of the H82 cells also significantly decreased H82 attachment, whereas the addition of antibodies to E-selectin, P-selectin, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), neural cell adhesion molecule (NCAM), sialyl-Lewis(x), fibronectin (Fn), alpha(v)-integrin, alpha3-integrin, alpha4-integrin, or alpha5-integrin had no effect on SCLC attachment. In summary, the TNF-alpha-mediated increase in SCLC attachment to ECs appears to be mediated by the activation of EC PKC and TK pathways as well as through effects on the function or expression of EC Vn and beta1 integrin.     

Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumor necrosis factor-alpha in endothelial cells

Oxidative signals play an important role in the regulation of endothelial cell adhesion molecule expression. Small GTP-binding protein Rac1 is activated by various proinflammatory substances and regulates superoxide generation in endothelial cells. In the present study, we demonstrate that adenoviral-mediated expression of dominant negative N17Rac1 (Ad.N17Rac1) suppresses tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin gene expression in a dose-dependent manner. Ad.N17Rac1 did not inhibit TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) binding activity or inhibitor of NF-kappaB-alpha degradation. In contrast, Ad.N17Rac1 inhibited TNF-alpha-induced NF-kappaB-driven HIV(kappaB)(4)-CAT and p288VCAM-Luc promoter activity, suggesting that N17Rac1 inhibits TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 through suppressing NF-kappaB-mediated transactivation. In addition, expression of superoxide dismutase by adenovirus suppressed TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 mRNA accumulation. However, adenoviral-mediated expression of catalase only partially inhibited TNF-alpha-induced E-selectin gene expression and had no effect on VCAM-1 and ICAM-1 gene expression. These data suggest that Rac1 and superoxide play crucial roles in the regulation of expression of cell adhesion molecules in endothelial cells.     

In vitro effects of cyclosporin A on the expression of adhesion molecules on human umbilical vein endothelial cells.

BACKGROUND: Among the different factors playing crucial roles in endothelial cell activation, cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been reported to demonstrate profound effects on this cell type. It has been shown that the increased release of IFN-alpha/gamma and TNF-alpha causes structural and functional modulations of the endothelial cell. These cytokines participate in the recruitment and activation of the immune system. CsA is an immunosuppressive drug that is necessary at high levels in human recipients of vascularised xenografts. This drug could contribute to a prolonged graft survival by modulation of endothelial cell activation. METHODS: The present study deals with the effects of cyclosporin A on adhesion molecule expression (i.e. ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1 and the L-selectin ligand CD 34) on the surface of cytokine stimulated HUVECs. The in vitro model described herein mimics the stimulation of endothelial cells by cytokines as seen during inflammatory processes after transplantation. Therefore, HUVECs were activated either with TNF-alpha, IL-1beta or with a cytokine mixture consisting of those stimulants present at an elevated level in sera of patients during allograft rejection (i.e. IL-1beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma). RESULTS: The results obtained show that the immunosuppression of CsA is not only achieved by inhibiting lymphocyte proliferation, but also by decreasing the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process. CONCLUSION: Co-incubation of stimulated endothelial cells with a final CsA concentration of 5 microg/ml revealed a significant down-regulating influence on the surface expression of E-selectin and VCAM-1.