活化血小板诱导内皮细胞表达黏附分子

血管内皮细胞释放黏附分子 ,促使循环单核细胞、淋巴细胞向局部黏附聚集 ,由此引发的炎症反应可造成冠状动脉内不稳定斑块发生破裂 ,继发血小板活化与血栓形成、并导致急性心肌缺血事件的发生[1 ] 。新近研究发现 ,血小板在其活化的同时尚可表达炎性介质ＣＤ1 54分子[2 ] 。提示除了参与血栓形成外 ,活化血小板也有可能通过ＣＤ1 54 ＣＤ4 0 信号途径主动介导炎症反应 ,但这一假设尚未被证实。为此 ,我们对血小板活化与其表达ＣＤ1 54间的确切关系进行了探讨。一、材料与方法1 .血小板的制备及诱导活化 :静脉血取自健康献血者及志愿者 (1 6…     

肝硬化患者血小板活化状态与内皮细胞损伤测定的临床意义

目的　探讨血小板活化状态与内皮细胞损伤在肝硬化病理生理及其临床意义。方法　对 5 8例肝炎后肝硬化患者与 30名正常对照者采用双抗体夹心放射免疫法测定血浆血小板α 颗粒膜蛋白 (GMP 14 0 ) ,放射免疫法测定内皮素 1(ET 1)含量。结果　肝硬化患者血浆GMP 14 0、ET 1水平比对照组有明显的增高 (P <0 .0 1) ,并且随着肝硬化Child分级程度的加重 ,升高越来越明显。结论　肝硬化患者血浆GMP 14 0、ET 1的升高可反映体内血小板活化及血管内皮细胞损伤的程度 ,在导致微循环障碍中起重要作用     

活化血小板表达CD154及其对单核细胞与内皮细胞黏附的促进作用

目的　探讨活化血小板通过表达CD15 4分子对单核细胞与内皮细胞黏附作用的影响。方法　以不同浓度二磷酸腺苷 (ADP) ,凝血酶体外诱导血小板活化 ,应用流式细胞术测定血小板CD6 2P、CD15 4表达水平 ,分析血小板表达CD15 4与其活化的关系 ;并将诱导或未经诱导的血小板与培养的人脐静脉内皮细胞 (HUVECs)共育 ,应用流式细胞术及ELISA法分别检测HUEVCs产生膜性及可溶性细胞间黏附分子 1(ICAM 1)的水平及变化 ,以孟加拉玫瑰红活细胞染色法测定单核细胞与HUVECs的黏附。结果　血小板活化程度与其CD15 4表达水平间呈显著正相关 (P <0 0 5 )。ADP(4μmol/L)或凝血酶 (1U/ml)诱导的血小板 ,均能显著增强单核细胞与HUVECs的黏附 ,并促进HUVECs产生膜性及可溶性ICAM 1(P <0 0 5、0 0 1) ,应用特异性CD15 4单抗后可阻断上述作用。静息血小板、单纯等量ADP或凝血酶 ,对HUVECs表达ICAM 1及单细胞与HUVECs的黏附均无影响 (P>0 0 5 )。结论　活化血小板可通过表达CD15 4促进单核细胞与内皮细胞的黏附 ,由此引发的炎症反应可能在动脉粥样硬化形成及不稳定斑块破裂方面发挥重要作用。     

内皮细胞的活化和损伤与急性呼吸窘迫综合征

简介内皮细胞的功能、内皮细胞活化的概念，及在急性呼吸窘迫综合征(ARDS)或其他危重病中内皮细胞活化与损伤标志物的变化，并探讨内皮细胞活化与损伤在ARDS发病机制中的作用。     

764-3对人血小板功能及血管内皮细胞抗凝作用的影响

研究了丹参的单体764-3对血小板α颗粒膜蛋白-140、血小板膜糖蛋白Ⅱ_b/Ⅲ_a复合物的表达和人脐静脉血管内皮细胞抗凝功能的影响。发现764-3能抑制血小板膜表面α颗粒膜蛋白-140和糖蛋白Ⅱ_b/Ⅲ_a复合物的分子表达,增加内皮细胞膜表面血栓调节蛋白的分子数和活性,这为阐明764-3的抗血栓作用机制提供了新的实验依据。     

丹酚酸B作用不同时间对高糖高脂内皮细胞凋亡的抑制作用

目的观察丹酚酸B抑制高糖高脂诱导的内皮细胞凋亡的作用。方法以糖基化终末产物(AGEs)和氧化低密度脂蛋白(ox-LDL)联合作用诱导人脐静脉内皮细胞凋亡,通过Hochest33258染色,观察并比较丹酚酸B及洛伐他汀作用不同时间(12、24、36、48、72h),抑制高糖高脂诱导的内皮细胞凋亡的效果。结果研究表明,洛伐他汀作用12、24h时,抑制内皮细胞凋亡效果明显,而丹酚酸B作用36、48h及72h抑制内皮细胞凋亡效果显著。结论与洛伐他汀相比,丹酚酸B抑制高糖高脂诱导的内皮细胞凋亡的作用发挥得较迟缓,但效果持久。     

起搏电极对血管内皮损伤与血小板活化的作用

通过追踪观察近 1年来安置起搏器病人血管内皮细胞受损和血小板活化的情况 ,探讨安置起搏器患者血小板活化的原因和机制。选择已安置起搏器的病窦综合征患者 12例 ,同期另选 12例未安置起搏器的病窦综合征病人作对照 ,两组年龄、性别、病情基本相匹配。检测血液中循环内皮细胞 (CEC)、血栓素B2 (TXB2 )、α 颗粒膜脂蛋白 140 (GMP 140 )的变化情况。检测前停抗凝、抗血小板及扩血管药物 5天。结果 :安置了起搏器的病人CEC数 (每 0 .9μL中的数量 )、TXB2 、GMP 140分别为 3 .0 7± 1.49个、37.78± 4.79ng/mL、2 6 2 .0 2± 36 .6 0pg/mL ;对照组则为 1.44±0 .99个、19.6 5± 6 .9ng/mL、16 3.0 2± 16 .0 7pg/mL ,均明显低于已安置起搏器者 (P <0 .0 0 1)。结论 :起搏电极机械损伤血管内皮 ,导致血管内皮脱落 ,内皮下胶原纤维暴露 ,可能是激活血小板的机制之一。     

肝硬变患者血小板活化状态与内皮细胞损伤测定的临床意义

目的探讨血小板活化状态与内皮细胞损伤在肝硬变病理生理及临床中的意义.方法对58例肝炎后肝硬变患者与30例正常对照者,采用双抗体夹心放射免疫法测定血浆血小板α-颗粒膜蛋白(GMP-),放射免疫法测定内皮素-1(ET-1)含量.结果肝硬变患者血浆GMP-140,ET-1水平比对照组有明显的增高(P＜0.01),并且随着肝硬变Child分级程度的加重,升高越来越明显.结论肝硬变患者血浆GMP-140,ET-1的升高可反映体内血小板活化及血管内皮细胞损伤的程度,在导致微循环障碍中起重要作用.     

丹酚酸B对老年性痴呆模型的保护作用

目的 探讨丹参提取物丹酚酸B(Sal B)对老年性痴呆(AD)模型大鼠学习记忆能力的影响及相关作用机制.方法 采用腹腔注射东莨菪碱建立AD模型大鼠,术后给予Sal B治疗.用Morris水迷宫检测大鼠的学习记忆能力;用试剂盒检测过氧化氢酶(CAT)、谷胱苷肽过氧化物酶(GSH-PX)活性;Western印迹检测凋亡相关蛋白表达.结果 经Sal B治疗后,AD模型大鼠学习记忆能力显著改善(P＜0.01).经Sal B治疗后,AD大鼠海马组织CAT、GSH-PX活力明显提高(P＜0.05).Sal B逆转AD模型大鼠凋亡相关蛋白表达(P＜0.01).结论 Sal B能改善AD大鼠学习和记忆能力,其机制可能与其促进自由基清除和抗凋亡有关.     

抗内皮细胞抗体介导的内皮细胞损伤机制

目的探讨抗内皮细胞抗体（antiendothelial cell antibody，AECA）对内皮细胞凋亡的影响及重组α-烯醇化酶对内皮细胞凋亡的阻断作用。方法以EA．hy926细胞（内皮细胞永生细胞）提取物蛋白为抗原，采用免疫印迹法检测并筛选47000-AECA阳性患者血清，用蛋白G亲和层析法纯化IgG，在体外诱导内皮细胞凋亡，观察重组α-烯醇化酶对凋亡的阻断作用。结果含47000-AECA IgG在体外可诱导内皮细胞凋亡，荧光染色可见明显的核形态变化及典型的凋亡小体；含47000-AECA系统性红斑狼疮患者的IgG作用于EA．hy926细胞24、48和72小时后，凋亡率均明显高于相同剂量正常人kG作用EA．hy926细胞的凋亡率；而经重组α-烯醇化酶预处理后EA．hy926细胞凋亡率则明显降低。含47000-AECA IgG作用于EA．hy926细胞8小时后，AnnexinV^＋细胞数明显增加，并随IgG作用时间和浓度的增加而升高；而经重组α-烯醇化酶预处理的含47000-AECA IgG作用于EA．hy926细胞后，AnnexinV^＋细胞有所减少。结论AECA在体外可诱导内皮细胞凋亡，引起内皮细胞损伤；重组α-烯醇化酶可部分阻断47000-AECA体外诱导内皮细胞凋亡的作用，α-烯醇化酶是AECA识别的自身抗原之一。     

别嘌呤醇对高糖诱导损伤的人脐静脉血管内皮细胞的保护作用

目的探讨别嘌呤醇对高糖损伤后的人脐静脉血管内皮细胞（HUVEC）的保护作用及其机制。方法以HUVEC为研究对象,体外培养至第三代,分为：①20mmol／L高糖损伤对照组;②别嘌呤醇保护组（浓度分别为0．1mmol／L、0．2mmol／L、0．3mmol／L）;③维生素C阳性对照组（浓度为100mg／L）。不同浓度别嘌呤醇及维生素C先与HUVEC孵育24h,再加入20mmol／L高糖诱导损伤48h,测定各组细胞上清液中一氧化氮（NO）、丙二醛（MDA）、超氧化物歧化酶（SOD）、细胞间黏附分子-1（ICAM-1）含量及细胞凋亡率。结果别嘌呤醇（0．1mmol／L、0．2mmol／L、0．3mmol／L）药物保护组的细胞凋亡率低于20mmol／L高糖组,其中以0．3mmol／L更为明显（P〈0．01）;细胞培养液中SOD、NO的量均较20mmol／L高糖组增高,其中以0．3mmd／L别嘌呤醇组更为明显（P〈0．01）。药物保护组细胞培养液中合成MDA、ICAM-1较波动性高糖组降低,且在一定浓度范围内（0．05-49．30mmol／L）呈浓度依赖性（P〈0．05）。结论①别嘌呤醇对高糖体外诱导的HUVEC损伤有保护作用,呈浓度依赖性。②别嘌呤醇对高糖体外诱导HUVEC损伤保护作用的机制包括抑制氧化应激、炎症反应及细胞凋亡。     

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E+ EMP. Markers of apoptosis were negative. In TTP patients, CD62E+ and CD31+/CD42b- EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E+ EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31+/42b- to CD62E+ EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.     

二甲双胍联合利拉鲁肽对棕榈酸诱导的内皮细胞氧化损伤具有协同保护作用

目的 探讨二甲双胍和利拉鲁肽在改善棕榈酸诱导的人脐静脉内皮细胞氧化损伤方面是否具有协同保护效应.方法 分离培养人脐静脉内皮细胞,应用不同浓度棕榈酸处理诱导细胞氧化损伤,观察不同浓度二甲双胍和（或）利拉鲁肽对细胞氧化损伤的影响.流式细胞仪检测细胞内的活性氧簇（ROS）水平,硝酸还原酶法检测上清一氧化氮（NO）的水平,组间比较采用单因素方差分析和Q检验.结果 与对照组相比,0.25和0.50 mmoL/L棕榈酸细胞内ROS水平显著升高[（125±17）％、（189±8）％比100％,P＜0.05],上清NO水平降低[（89.9±6.2）％、（79.8±4.8）％比100.0％,P＜0.05];二甲双胍（0.5 ～ 1.0 mmol/L）和利拉鲁肽（10 ～ 100 nmol/L）单独应用后,可使0.50 mmol/L棕榈酸所致的ROS产生增加和NO产生减低作用下降;低剂量的二甲双胍（0.1 mmol/L）或利拉鲁肽（3 nmoL/L）单独应用对0.5 mmol/L棕榈酸的作用均无明显的影响,但两者联合则可减低上述作用：两者联合组与棕榈酸组相比,ROS水平降低[（158±31）％比（250±27）％,P＜0.05],NO水平增加[（91.7±30.6）％比（82.3±5.0）％,P＜0.05].结论 二甲双胍和利拉鲁肽在改善棕榈酸诱导的内皮细胞氧化损伤方面具有协同效应.     

Inhibitory effect of melatonin on platelet activation induced by collagen and arachidonic acid

Abstract: Melatonin, an indolamine synthesized in the pineal gland, is known to have antiprostanoid activity. The inhibition of platelet aggregation induced by melatonin has been proposed to take place through the cyclooxygenase pathway. In the present study, we found that melatonin has a marked inhibitory effect on collagen, arachidonic acid (AA), adenosine diphosphate (ADP), epinephrine, and A23187-induced aggregation in platelet-rich plasma. On the other hand, using metrizamide-filtered platelets resuspended in Tyrode's buffer, melatonin fails to suppress AA-induced platelet aggregation and ¹⁴C-5-HT release. Under the same conditions, melatonin inhibits collagen-induced platelet activation; however, the addition of threshold doses of AA (0.3 mM) abrogates this effect. These studies suggest that melatonin also inhibits platelet function at a stage preceding the cyclooxygenase-dependent pathway.     

Synergism between melatonin and atorvastatin against endothelial cell damage induced by lipopolysaccharide

The beneficial effects of atorvastatin are based on both cholesterol-dependent and independent mechanisms. The latter probably include the ability of the estatin to enhance the expression of endothelial nitric oxide synthase (eNOS) and to cause a vasodilatation. In turn, the antioxidant and anti-inflammatory actions of melatonin are related to its vascular protection. In the present study, we investigated the efficacy of the combination of melatonin plus atorvastatin against endothelial cell damage induced by inflammation and oxidative stress injury. Human umbilical vein endothelial cells (HUVEC) were cultured with bacterial lipopolysaccharide (LPS) in the presence or absence of melatonin and/or atorvastatin. LPS inhibited eNOS mRNA and protein expression, which was reversed by atorvastatin and, to a lesser extent, by melatonin. Together, melatonin + atorvastatin induced higher eNOS protein expression than either compound alone. Melatonin, but not atorvastatin, reduced free radical generation, lipid peroxidation, and interleukin-6 levels induced by LPS. In the presence of atorvastatin, the effects of melatonin were maintained or even improved. These data suggest that melatonin improves the beneficial effects of atorvastatin and reduces its side effects in endothelial cells during inflammation and under conditions of oxidative stress.     

Signalling pathways involved in retinal endothelial cell proliferation induced by advanced glycation end products: inhibitory effect of gliclazide

Aim:  We have previously demonstrated that advanced glycation end products (AGEs) stimulate bovine retinal endothelial cell (BREC) proliferation through induction of vascular endothelial growth factor (VEGF) production by these cells. We have also shown that gliclazide, a sulfonylurea which decreases oxidative stress, inhibits this effect. The aim of the present study was to characterize the signalling pathways involved in AGE-induced BREC proliferation and VEGF production and mediating the inhibitory effect of gliclazide on these biological events.
Methods:  BRECs were treated or not treated with AGEs in the presence or absence of gliclazide, antioxidants, protein kinase C (PKC), mitogen-activated protein kinase (MAPK) or nuclear factor-κB (NF-κB) inhibitors. BREC proliferation was assessed by measuring [³H]-thymidine incorporation into DNA. Activation of PKC, MAPK and NF-κB signal transduction pathways and determination of VEGF expression were assessed by Western blot analysis using specific antibodies. MAPK activity was also determined by an in vitro kinase assay.
Results:  Treatment of BRECs with AGEs significantly increased cell proliferation and VEGF expression. AGEs induced PKC-β translocation, extracellular signal-regulated protein kinase 1/2 and NF-κB activation in these cells. Pharmacological inhibition of these signalling pathways abolished AGE effects on cell proliferation and VEGF expression. Exposure of BRECs to gliclazide or antioxidants such as vitamin E or N -acetyl- l -cysteine resulted in a significant decrease in AGE-induced activation of PKC-, MAPK- and NF-κB-signalling pathways.
Conclusions:  Our results demonstrate the involvement of PKC, MAPK and NF-κB in AGE-induced BREC proliferation and VEGF expression. Gliclazide inhibits BREC proliferation by interfering with these intracellular signal transduction pathways.