细胞分化抗原40与细胞分化抗原40配体相互作用对内皮细胞明胶酶及其抑制物表达的影响

目的 :探讨细胞分化抗原 (CD ) 40与CD40配体 (CD40L )相互作用对内皮细胞明胶酶 (MMP2 和MMP9)及其抑制物 (TIMP1和TIMP2 )表达的影响。　　方法 :建立内皮细胞与巨噬细胞膜共孵育体系 (细胞比例 1∶1) ,观察内皮细胞明胶酶及其抑制物活性、蛋白表达的变化 ,以及阻断CD40与CD40L相互作用对上述效应的影响。　　结果 :共孵育 18小时后内皮细胞MMP9和活化型MMP2 活性分别增强 2 3 8和 3 40倍 (P <0 0 1) ;阻断CD40L的作用可抑制此效应分别达 60 1%和 5 3 6% (P <0 0 1)。同时 ,活化型MMP2 与酶原型MMP2 活性之比由 0 2 5升至0 47(P <0 0 1) ,阻断CD40L的作用后降至 0 3 7(P <0 0 1)。明胶酶蛋白表达与酶活性的变化是一致的。而TIMP1和TIMP2 活性和蛋白表达不受巨噬细胞膜或抗CD40L抗体的明显影响。　　结论 :巨噬细胞通过CD40与CD40L相互作用增强内皮细胞明胶酶活性及蛋白表达 ,可能是动脉粥样硬化斑块易于破裂的原因之一。     

活化Toll样受体4诱导静脉血管内皮细胞介导非耐受性炎症反应

目的:通过分析活化Toll样受体4(TLR4)对人脐静脉内皮细胞分泌炎性细胞因子的影响,探讨静脉内皮细胞在炎症反应中的作用.方法:逆转录聚合酶链式反应检测TLR4和细胞因子信使核糖核酸(mRNA)在人脐静脉内皮细胞中的表达,流式细胞分析检测TLR4及其辅助受体CD14的蛋白表达水平,酶联免疫吸附试验检测细胞培养上清中细胞因子的表达水平.结果:TLB4配体脂多糖诱导了人脐静脉内皮细胞中TLB4的基因和蛋白表达上调以及CD14的蛋白表达上调.活化TLR4显著上调人脐静脉内皮细胞中炎性细胞因子白细胞介素(IL)-1β和IL-8的基因表达以及IL-6和肿瘤坏死因子(TNF)-α的蛋白表达,并呈剂量依赖关系.活化TLR4诱导的细胞因子上调依赖NF-кB信号传导途径,信号传导阻滞剂能抑制细胞因子的表达上调.而且初次活化后,TLR4的再次活化仍然诱导高水平的炎性细胞因子.结论:活化TLR4诱导静脉内皮细胞分泌炎性细胞因子,介导非耐受性炎症反应,提示静脉内皮细胞是重要的炎症效应细胞.     

阿托伐他汀对缺氧再复氧脐静脉内皮细胞CD40配体表达的影响

目的研究不同浓度阿托伐他汀对缺氧/再复氧诱导的人脐静脉内皮细胞CD40配体表达水平的影响,探讨阿托伐他汀对内皮细胞损伤的保护机制。方法采用培养的人脐静脉内皮细胞,给予缺氧1 h,再复氧3 h处理,建立缺氧/再复氧损伤模型,给予不同浓度阿托伐他汀预处理,通过流式细胞仪测定脐静脉内皮细胞CD40配体表达水平的变化。结果缺氧/再复氧处理后脐静脉内皮细胞CD40配体表达水平显著增高,阿托伐他汀可下调缺氧/再复氧所诱导的细胞CD40配体的表达,并随其药物浓度的增高(0.1、1、5、10μmol/L),CD40配体的表达逐渐下降。结论他汀类药物可以下调内皮细胞CD40配体的表达,并随其浓度的增加下调作用更明显,从而发挥其调脂外的对缺氧/再复氧内皮保护和抗炎等作用。     

白藜芦醇对内皮细胞CD40途径的影响

目的：观察白藜芦醇对内皮细胞CD40途径活化后CD40和E-选择素表达的影响。方法：白藜芦醇（10μmol／L）预孵育人脐静脉内皮细胞（HUVEC）后,以可溶性CD40配体（sCD40L,10μg／m1）刺激,流式细胞术检测内皮细胞E-选择素和CD40分子的表达,半定量逆转录聚合酶链式反应（RT—PCR）检测E-选择素和CD40基因的转录。结果：sCD40L诱导内皮细胞E-选择素和CD40基因的显著转录和表达（P均〈0．01）,白藜芦醇显著抑制E-选择素和CD40基因的转录和表达（P〈0．05～〈0．01）。结论：结果提示白藜芦醇可能通过抑制CD40途径发挥抗动脉粥样硬化作用。     

血小板因子4促进人脐静脉内皮细胞Toll样受体2的表达

目的 探讨血小板因子4对人脐静脉内皮细胞Toll样受体2表达的影响.方法 采用人重组细胞因子血小板因子4刺激人脐静脉内皮细胞株ECV304,通过RT-PCR和Western Blotting分别检测ECV304中Toll样受体2 mRNA和蛋白表达.细胞免疫组织化学法检测血小板因子4刺激后ECV304中Toll样受体2的表达量.结果 血小板因子4刺激人脐静脉内皮细胞株后能促进Toll样受体2 mRNA和蛋白表达.与空白组相比,100μg/L血小板因子4处理人脐静脉内皮细胞株能在基因及蛋白水平促进其Toll样受体2表达并呈现一定的时间依赖性(n=5,P<0.05),且肝素不能抑制血小板因子4的这种作用.细胞免疫组织化学法也显示与空白组(0.001 385±0.000 953)相比,血小板因子4处理后人脐静脉内皮细胞Toll样受体2表达显著增多(0.060 399±0.020 998,P<0.05).结论 血小板因子4促进人脐静脉内皮细胞株表达Toll样受体2,此效应可能与血小板在动脉粥样硬化中的作用有关.     

血管紧张素(1-7)对CD40及CD40配体信号通路及活化黏附分子的抑制作用

目的观察血管紧张素1-7[Ang-(1-7)]对血管紧张素Ⅱ(AngⅡ)诱导的人脐静脉内皮细胞中细胞间黏附分子1(ICAM-1)和血管细胞黏附分子1(VCAM-1)表达的影响及作用机制是否与CD40及CD40配体(CD40L)通路有关。方法人脐静脉内皮细胞培养后,分6组:对照组;AngⅡ组;低、中和高剂量组[Ang-(1-7)10、100和1000nmol/L预处理];阻断剂组(A-779预处理)。用RT-PCR法检测ICAM-1、VCAM-1和CD40、CD40L mRNA表达。结果与对照组比较,AngⅡ组ICAM-1、VCAM-1和CD40、CD40L mRNA表达显著升高(P0.05);与AngⅡ组比较,低、中和高剂量组ICAM-1、VCAM-1和CD40、CD40L mRNA的表达逐渐下降(P0.05),其中ICAM-1、VCAM-1mRNA表达分别下降25%、58%、69%和30%、53%、62%;CD40、CD40L mRNA表达分别下降35%、48%、61%和26%、54%、68%;阻断剂组与AngⅡ组上述指标比较无显著差异(P0.05)。结论 Ang-(1-7)可以抑制炎症通路CD40/CD40L活化,进而下调黏附分子ICAM-1、VCAM-1的表达;Ang-(1-7)的抗炎机制是首先与Mas受体结合,进而发挥抑制CD40/CD40L通路的作用。     

CD40 mRNA表达与胃癌发生发展及预后关系的探讨

肿瘤坏死因子受体(TNFR)超家族成员CD40及其配体CD40L是特异性免疫反应系统中重要的一对共刺激分子,能介导细胞凋亡,在抗肿瘤细胞方面起重要作用.近年来的研究发现,CD40分子在某些肿瘤组织中异常表达,且与肿瘤侵袭、转移等生物学行为密切相关.本研究采用原位杂交技术检测64例胃癌组织和16例正常胃黏膜组织中CD40 mRNA的表达,探讨CD40 mRNA在胃癌中的表达规律及其与临床的关系.     

川芎嗪抑制凝血酶诱导血管内皮细胞组织因子表达的机制

目的 以前的研究已证实川芎嗪对凝血酶诱导人脐静脉内皮细胞株表达组织因子具有抑制效应.本实验进一步探讨一氧化氮及核因子KB途径在其中所发挥的作用机制.方法 人脐静脉内皮细胞细胞培养采用RPMI-1640完全培养基;一期凝固法测总促凝活性;组织因子mRNA采用逆转录聚合酶链式反应方法检测;免疫组织化学染色用于研究核因子KB的移位.结果 一氧化氮合酶途径阻断剂硝基左旋精氨酸甲基乙酯单独和人脐静脉内皮细胞细胞孵育时对细胞表达组织因子mRNA和总促凝活性没有明显的影响(P>0.05);硝基左旋精氨酸甲基乙酯、川芎嗪和凝血酶三者共同孵育时,川芎嗪抑制凝血酶诱导人脐静脉内皮细胞细胞组织因子表达的作用被取消(P<0.05).免疫组织化学染色显示人脐静脉内皮细胞细胞经凝血酶处理45 min后,细胞核内棕黄色着色明显,但人脐静脉内皮细胞细胞经川芎嗪处理15 min后,再加入凝血酶作用45 min,核内棕黄色着色则明显减少.结论 一氧化氮途径参与了川芎嗪抑制凝血酶诱导血管内皮细胞表达组织因子的作用;川芎嗪能够通过影响核因子KB的活化来抑制组织因子的表达.     

NF-κB在氧化型低密度脂蛋白引起人脐静脉内皮细胞表达CD40中的作用

目的研究氧化低密度脂蛋白(ox-LDL)对人脐静脉内皮细胞表达分化抗原CD40中核因子κB(NF-κB)的作用,进一步探讨卡托普利可能的抗动脉硬化作用。方法在ox-LDL作用人脐静脉内皮细胞前预先用卡托普利、NF-κB阻断剂(PDTC)、卡托普利+一氧化氮合酶阻断剂(L-NAME)、卡托普利+PDTC作用后,应用流式细胞技术检测细胞表面CD40的表达。结果预先加入卡托普利、PDTC人脐静脉内皮细胞的CD40的表达低于ox-LDL组(P<0.05),卡托普利组内皮细胞CD40的表达值低于卡托普利+PDTC组和卡托普利+NAME组,组间相比差异显著(P<0.001)。结论ox-LDL通过NF-κB途径激活了CD40的表达,卡托普利通过一氧化氮(NO)途径及NF-κB的转录使ox-LDL引起的内皮细胞CD40的表达下降,从而具有抗动脉硬化作用。     

U937源性泡沫细胞形成中组织因子途径抑制物2的表达定位及变化规律

目的研究U937源性泡沫细胞形成过程中组织因子途径抑制物2的表达定位及变化规律。方法采用沉淀法提取人血浆低密度脂蛋白,硫酸铜氧化制备氧化型低密度脂蛋白,与U937单核细胞共同孵育,建立泡沫细胞模型,同时体外培养人脐静脉内皮细胞,加入U937泡沫细胞中共同培养,观察内皮细胞对泡沫细胞形成的影响。采用双重细胞免疫荧光定位组织因子途径抑制物2蛋白,时间免疫分辨荧光定量组织因子途径抑制物2蛋白,逆转录聚合酶链反应和荧光定量聚合酶链反应检测泡沫细胞内组织因子途径抑制物2基因的动态变化。结果组织因子途径抑制物2主要定位于U937单核细胞胞质中,与组织因子有相似的分布。氧化型低密度脂蛋白作用于U937细胞6h后组织因子途径抑制物2蛋白和基因表达水平持续增高,6h达至峰值而后表达持续降低。氧化型低密度脂蛋白作用于人脐静脉内皮细胞后组织因子途径抑制物2蛋白表达增加,24h时达至峰值。加入内皮细胞可改善氧化型低密度脂蛋白抑制U937细胞表达组织因子途径抑制物2的作用。结论U937泡沫细胞形成过程中,体外培养的人源性单核细株胞U937上存在组织因子途径抑制物2的表达抑制,而与人脐静脉内皮细胞共同培养可改善这种异常。     

Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression

Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell-induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D(3)-differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti-P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator-stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti-P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.     

Platelet-CD40 ligand interaction with melanoma cell and monocyte CD40 enhances cellular procoagulant activity.

Platelet-tumor cell interactions are believed to be important in tumor metastasis. Tumor cell tissue factor (TF) expression enhances metastasis and angiogenesis, and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell-platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression by ligation to CD40. We investigated the effect of platelet-derived CD40L on the TF activity of human CD40-positive melanoma cells and monocytes by incubating supernatants from activated or resting platelets with tumor cells or monocytes, and by bringing resting or activated platelets into close apposition with tumor cell monolayers. CD40L was present on the surface of activated (but not resting) platelets and was also released following platelet activation. Both recombinant soluble CD40L (rsCD40L) and activated platelet supernatants increased procoagulant activity (PCA) and TF antigen in tumor cells and monocytes. The increase in TF activity induced by both rsCD40L and activated platelet supernatants was inhibited by anti-CD40L antibody. Furthermore, contact of activated platelets with tumor cells increased cellular PCA, and this effect was also inhibited by anti-CD40L. In malignancy, the increase in cellular TF activity via CD40 (tumor cell)-CD40L (platelet) interaction may possibly enhance intravascular coagulation and hematogenous metastasis.     

Effects of ethanol on the properties of platelets and endothelial cells in model experiments

AIM: To investigate effects of ethanol on activity markers of atherosclerosis in an in vitro endothelial cell model. METHODS: After 24 h incubation with ethanol (0.0095%), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide, and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets, and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urokinase plasminogen activator receptor (uPAR), and membrane-type 1 matrix metalloproteinase (MT1-MMP) on endothelial cells were measured by flow cytometry. RESULTS: The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through pre-incubation with ethanol (P<0.05). Furthermore, platelets in direct contact with ethanol and with endothelial cells pre-incubated in ethanol showed a significant reduction in their CD40L expression (P<0.05). Ethanol had no significant effect on ICAM-1 and MT1-MMP expression on endothelial cells. CONCLUSION: Ethanol directly attenuates platelet activation and has significant endothelial cell-mediated effects on selected markers of atherosclerosis in vitro . These findings underline possible protective effects of ethanol on atherosclerosis.     

晚期糖基化终产物诱导内皮细胞黏附连接改变及其机制

目的观察不同浓度和时间修饰的糖基化人血清白蛋白作用下,人脐静脉内皮细胞内黏附连接蛋白钙黏着蛋白的形态结构变化,并初步探讨其机制。方法原代培养人脐静脉内皮细胞,分别用不同浓度和时间修饰的糖基化人血清白蛋白处理,用免疫荧光染色法、激光共聚焦显微镜观察钙黏着蛋白在内皮细胞的形态和分布变化。分别用可溶性的修饰的糖基化人血清白蛋白受体的抗体、丝裂原活化蛋白激酶抑制剂或转染重组腺病毒突变体转染预处理后再观察晚期糖基化终产物修饰的人血清白蛋白对内皮细胞形态的影响。结果修饰的糖基化人血清白蛋白以时间和剂量依赖方式引起内皮细胞黏附连接钙黏着蛋白形态结构的改变;丝裂原活化蛋白激酶通路抑制剂(SB203580、PD98059、SP600125)和Rho激酶抑制剂Y27632均可减轻晚期糖基化终产物对钙黏蛋白的影响;转染显性失活的细胞外信号调节激酶上游激酶MEK1和p38丝裂原活化蛋白激酶上游激酶MKK6b及p38显性失活的p38α和p38β重组腺病毒突变体,均可减轻晚期糖基化终产物对钙黏蛋白形态结构的影响,而转染组成性激活的MEK1和MKK6b的重组腺病毒本身即可引起钙黏蛋白形态结构的变化。结论晚期糖基化终产物刺激可以引起钙黏蛋白分布和形态的变化,这一作用可能是丝裂原活化蛋白激酶通路细胞外信号调节激酶、p38丝裂原活化蛋白激酶信号通路和c-jun氨基末端激酶(JNK)/应激激活的蛋白激酶(SAPK)介导的,Rho激酶可能参与此过程。     

CD40-ligand-dependent induction of COX-2 gene expression in endothelial cells by activated platelets: inhibitory effects of atorvastatin.

Increasing evidence shows the importance of platelet-endothelial cell interactions in the progression of atherosclerosis. Platelets contribute to coronary events both as major components of thrombi and as a triggering factor in inflammation that leads to plaque vulnerability. Recent data suggest that statins, besides their lipid-lowering properties, exert pleiotropic effects that may be beneficial in atherosclerosis. Whether activated platelets influence cyclooxygenase-2 (COX-2) expression in human umbilical vein endothelial cells (HUVEC), the effect of atorvastatin, and possible mechanisms were investigated. COX-2 gene expression in HUVEC was studied using real-time polymerase chain reaction. CD40 ligand surface expression of platelets was tested by fluorescence-activated cell sorting analyses. Activated platelets significantly up-regulated COX-2 gene expression in HUVEC. Co-incubation of platelets with atorvastatin was shown to reverse this up-regulation via reduction of CD40 ligand surface expression on platelets. Data suggest that atorvastatin influences CD40-CD40-ligand-dependent platelet-endothelial interaction and that this influence affects platelet-induced COX-2 expression in HUVEC.     

脂质体介导血管内皮生长因子基因在内皮细胞的稳定表达

建立稳定表达血管内皮生长因子的人脐静脉内皮细胞系 ,为其在组织化工程血管的应用奠定基础。将真核表达载体PCD2 VEGF1 2 1 用阳离子脂质体介导 ,转染人脐静脉内皮细胞系细胞 ,G4 18筛选 ,获得G4 18抗性单克隆细胞 ,扩增后分别用逆转录聚合酶链反应、免疫组织化学及血管通透性实验检测血管内皮生长因子的转录、蛋白质的表达及其生物学活性。结果发现 ,逆转录聚合酶链反应检测出了转录血管内皮生长因子的稳定转染细胞克隆 ,该单克隆细胞的免疫组织化学检测血管内皮生长因子蛋白质表达呈阳性 ,血管通透性实验和细胞生长实验发现其表达产物具有生物学活性 ,而作为对照的转空白质粒细胞和未转染细胞上述实验结果皆为阴性。结果表明 ,该方法成功建立了稳定表达血管内皮生长因子的人脐静脉内皮细胞系单克隆细胞     

氧化型低密度脂蛋白和抗氧化剂对人血管内皮细胞钙转运的影响

为探讨氧化型低密度脂蛋白和抗氧化剂对内皮细胞钙转运功能的影响 ,揭示氧化型低密度脂蛋白毒性作用的分子机制和预防措施 ,采用体外培养的人脐静脉内皮细胞 ,单独给予氧化型低密度脂蛋白及同时给予氧化型低密度脂蛋白和抗氧化剂维生素E ,分别测定细胞钙摄取和释放功能 ,并测定细胞及上清液乳酸脱氢酶活性 ,计算细胞死亡率。结果发现 ,氧化型低密度脂蛋白呈剂量依赖性增加细胞死亡率 ,同时刺激内皮细胞钙摄取增加 ,对钙释放无影响 ,维生素E可抑制氧化型低密度脂蛋白的上述作用。结果提示 ,氧化型低密度脂蛋白刺激内皮细胞钙摄取可能是其致内皮细胞损伤的机制 ,维生素E通过抑制此作用而达到细胞保护的效应。     

Platelets trigger a CD40-dependent inflammatory response in the microvasculature of inflammatory bowel disease patients

BACKGROUND & AIMS: Platelets circulate in an activated state in patients with inflammatory bowel disease (IBD), but their role in the pathogenesis of IBD is unclear. The recent demonstration that activated platelets express CD40 ligand (L) provides a mechanism of interaction with CD40-positive endothelial cells, inducing them to produce proinflammatory mediators. We investigated whether platelets from patients with IBD express enhanced levels of CD40L and induce human intestinal microvascular endothelial cells (HIMEC) to up-regulate cell adhesion molecule (CAM) expression and secrete chemokines. METHODS: CD40L expression was assessed in resting and thrombin-activated platelets by flow cytometry and in mucosal microthrombi by confocal microscopy. Platelet-HIMEC cocultures were used to study CAM up-regulation, and interleukin (IL)-8 and RANTES production by HIMEC. RESULTS: IBD platelets expressed significantly higher CD40L levels than those of healthy subjects, and CD40L-positive platelets were detected in IBD-involved mucosa. Activated platelets up-regulated expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 as well as production of interleukin 8 by HIMEC in a CD40-dependent fashion. High levels of RANTES were present in platelet-HIMEC cocultures and platelets were identified as the source of this chemokine, which mediated T-cell adhesion to HIMEC. CONCLUSIONS: These results show that platelets can actively contribute to mucosal inflammation and represent a previously unrecognized component of IBD pathogenesis.