改良墨汁灌注法与von Willebrandfactor免疫荧光法在大鼠视网膜微血管形态显示中的对比研究

目的:比较改良墨汁灌注法与von Willebrand factor(vWf)免疫荧光法在大鼠视网膜微血管形态显示方面的异同.方法:成年健康SD大鼠随机分成vWf免疫荧光显色组和改良墨汁灌注组.改良墨汁灌注组分两步灌注明胶墨汁40 ml.vWf免疫荧光显色组常规灌注生理盐水40 ml.视网膜行铺片和切片,观察vWf免疫荧光法和改良的墨汁灌注法显示的微血管形态;两组视网膜切片还进行NeuN、Parvalbumin和GFAP的免疫组织化学显色.结果:vWf免疫荧光法能充分显示视网膜铺片周嗣部的微血管,但对铺片中央部和切片的微血管显示不良;改良墨汁灌注法能充分显示大鼠视网膜铺片和切片的微血管,用此方法灌注的视网膜切片的NeuN、Parvalbumin和GFAP免疫组织化学效果均与正常视网膜相似.结论:改良墨汁灌注法在大鼠视网膜微血管形态显示中优于vWf免疫荧光法,且能在同一切片上准确显示血管与神经元/胶质细胞的空间关系.     

脑数字减影血管造影术对血浆ET-1和vWF水平的影响

目的: 探讨脑数字减影血管造影术(DSA) 对血浆内皮素-1(ET-1)及von Willebrand因子(vWF)的水平的影响。方法: 用ELISA法测定连续收集的108例缺血性脑血管病患者脑DSA术前、术后即刻、术后24 h血浆ET-1和vWF的含量。结果: 患者脑DSA后血浆ET-1水平均明显升高,ET-1在术后即刻有明显升高,为(550.19±108.06)ng/L),术后24 h有所下降,为(517.26±111.84)ng/L,但仍高于术前水平,3个时点血浆ET-1水平有明显差异。血浆vWF水平术后未观察到明显升高。结论: 术后ET-1的持续升高提示脑DSA可能对血管内皮细胞造成一定程度的损伤。     

急性呼吸窘迫综合征患者血液中von Willebrand因子含量的变化

目的：观察21例急性呼吸窘迫综合征（acute respiratory distress syndrome, ARDS）患者发病早期血液中血管性假血友病因子（von Willebrand factor, vWF）含量的变化，考察其对ARDS早期诊断价值。方法：21例ARDS患者第1 d，30例急性左心衰竭、慢性阻塞性肺病（chronic obstructive pulmonary disease, COPD）肺部感染患者及正常对照同期抽血测vWF。结果：ARDS患者血vWF为325%±63%，急性左心衰竭患者血vWF为107%±42%，COPD肺部感染患者血vWF为103%±48%，正常对照者为92%±36%。ARDS组明显高于各对照组（P<0.01）。结论：ARDS患者在发病早期血vWF即明显升高，提示血vWF含量对ARDS的早期诊断有参考价值。     

不稳定性心绞痛患者血管性血友病因子与血管细胞间粘附分子水平的变化

目的：探讨老年不稳定性心绞痛（UA）患者血管性血友病因子（vWF）与可溶性血管细胞间粘附分子-1（sVCAM-1）水平变化及其与心肌缺血的关系。 方法： 采用酶联免疫吸附法（ELISA），检测了50例健康人和73例UA（自发性心绞痛27例，心肌梗死后心绞痛25例，恶化劳力性心绞痛21例）患者血浆vWF、sVCAM-1浓度的变化。 结果： ①UA患者血浆vWF水平、sVCAM-1浓度［（2.47±0.88）%、（1.92±0.51）%、（961±58）μg/L、（692±73）μg/L］，明显高于对照组［（572±58）μg/L、（0.96±0.14）%］（P＜0.01）；②心绞痛发作时vWF、sVCAM-1浓度增高较缓解后更明显（P＜0.01）；③心绞痛发作时和缓解后，sVCAM-1与vWF呈正相关（r=0.785，r=0.674，P＜0.01 ）；④不同类型的心绞痛发作时和缓解后sVCAM-1、vWF浓度差异亦具有显著性（P＜0.01）；⑤自发性心绞痛患者vWF、sVCAM-1增高较心肌梗死后和恶化劳力性心绞痛更明显。 结论： 急性心肌缺血与vWF和sVCAM-1异常有一定关系。     

静脉留置针对兔耳缘静脉血栓形成及血浆组织因子与血管性假血友病因子水平的影响

目的:观察静脉留置针于兔耳缘静脉置管不同时间的血栓形成情况,同时检测血浆组织因子(TF)和血管性假血友病因子(vWF)水平变化,探讨留置针致静脉血栓形成的机制。方法:50只新西兰兔随机分成5组:即正常对照组、置管1天组、置管3天组、置管5天组、置管7天组。取兔右耳外侧耳缘静脉留置针置管输液,每日经留置针输入生理盐水20ml。于置管第2天、第4天、第6天和第8天分别取各组兔耳输液血管组织行HE染色,观察病理形态学变化和血管内血栓形成情况;同时经颈静脉取血,ELISA检测血浆TF和vWF水平变化。结果:正常对照组兔耳缘静脉血管壁完整,无血栓形成,但置管3天组、5天组、7天组血管壁完整性出现不同程度损害,有明显血栓形成,血栓形成率分别为30%、70%、80%。与正常对照组相比,置管3天组、5天组、7天组血浆TF和vWF水平显著升高(p0.05)。结论:静脉留置针置管3天以上可致部分实验动物血栓形成,其机制可能与其血浆TF和vWF水平升高有关。     

Roles of claudin-5 and von Willebrand factor in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that affects approximately 1% of the world’s population. The pathogenesis of RA is not understood fully. It is assumed that endothelial function is associated with the proinflammatory state of RA. Endothelial dysfunction/activation reflects the increased level of von Willebrand factor (vWF) and a shift toward prothrombotic activity of the endothelium. The present study was performed to investigate the possible relationships between vWF and claudin-5 and the level of disease activity in patients with RA. The study population was divided into four groups according to the disease activity score in 28 joints (DAS28): remission group (RG), 18 patients (DAS28 < 2.6); low disease activity group (LDAG), 23 patients (DAS28 > 2.6-3.2); moderate disease activity (MDAG), 23 patients (DAS28 > 3.2-5.1); high disease activity group (HDAG), 14 patients (DAS28 > 5.1); and control group (CG), 10 healthy subjects. Claudin-5 and vWF assessment were derived from serum samples gathered from the patients known to have RF and anti-CCP titers in the normal ranges. A high positive association of claudin-5 and vWF with the MDAG was observed (P < 0.001). The results of our study indicated that the relationship between vWF and claudin-5, which are indicators of endothelial cell dysfunction and tight junction activity, may be a predictor of disease activity. Further studies are required to investigate these pathways to shed light on the roles of claudin-5 and vWF in the progression of inflammation and other vascular conditions.     

Soluble cell adhesion molecules and von Willebrand factor in children with Kawasaki disease.

Fifty-nine children with acute Kawasaki disease (KD), a childhood vasculitis, were compared with 35 children with fever due to infection and 48 healthy children. Levels of soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1) in the healthy children were double those found in adults. All three soluble cell adhesion molecules and von Willebrand factor (vWF) were higher in the children with KD than in the healthy children, but only sE-selectin, a marker for activated endothelial cells, and sICAM-1 were higher than in the febrile children. The high levels of vWF in KD appear to reflect the prominent acute-phase reaction. This information can help us to understand further the complex interactions between cytokines, circulating inflammatory cells and the vascular endothelium, and may lead to new therapeutic avenues in KD and other inflammatory diseases and vasculitides.     

Differing microvasculature in the two major types of gastric carcinoma: a conventional,ultrastructural and ultrastructural immunolocalization study of von Willebrand factor

Summary The microvasculature of the stroma of human gastric carcinoma was studied by immuno-electron microscopy for factor VIII/von Willebrand factor (vWF) and conventional electron microscopy. In differentiated type (intestinal) gastric carcinoma (9 cases), capillaries were distributed more densely around carcinoma cell nests. vWF was localized in endothelial cells and neighbouring stroma. Ultrastructurally, capillary endothelial cells showed considerable hypertrophic changes with well-developed rough endoplasmic reticulum (rER). vWF was localized in well-developed rER, granules, Weibel-Palade bodies (WPB), in the vascular lumen as clusters, and diffusely deposited in the subendothelium. This indicates that endothelial cells in this group are transformed into a state of active protein production. In undifferentiated type (diffuse) gastric carcinoma (12 cases), capillaries were uniformly distributed and endothelial hypertrophic changes were less remarkable. vWF was localized in WPB, scanty rER and subendothelial matrix. Solid capillary buds were observed in both types; they were composed of a solid strand of endothelial cells without a visible lumen. Our results reveal that the microvasculature in tumour stroma differs significantly according to its histological type.     

血管性血友病因子裂解酶金属蛋白酶域的克隆和表达

目的:克隆和表达血管性血友病因子裂解酶(vWF-cp)的金属蛋白酶域。方法:应用PCR从含有人vWF-cp金属蛋白酶域的质粒中扩增出该区域,克隆至pUC18载体后行序列分析,并将其重组于带有6×HisTag的融合蛋白表达载体pET28a(+)中,在大肠杆菌DE3／plySs中诱导表达。融合蛋白经Ni-NTA柱纯化后,采用Westernblot印迹鉴定。结果:PCR扩增得到658bp的vWF-cp金属蛋白酶域的基因片段,测序结果与GenBank序列一致。重组表达质粒经异丙基硫代-β-D-半乳糖苷(IPTG)诱导5h后表达的融合蛋白占菌体总蛋白的28％,进一步纯化后其纯度在95％以上。结论:在大肠杆菌中高效表达了人vWF-cp的金属蛋白酶结构域,为深入研究vWF-cp结构与功能的关系奠定了基础。     

糖尿病视网膜病变与血管性血友病因子和纤维蛋白原的关系

目的 :探讨糖尿病视网膜病变 (DR)与纤维蛋白原 (FIB)及vonWillebrand因子 (vWF)水平的关系及其临床意义。方法 :2型糖尿病人 10 4例根据病史及是否合并糖尿病微血管病变进行如下分组 :无并发症组 5 3例 (男 2 6例 ,女 2 7例 ) ,合并糖尿病视网膜病变组 5 1例(男 2 5例 ,女 2 7例 ) ,正常对照组 5 6例 (男 2 8例 ,女 2 8例 )。三组分别采用硫酸胺比浊法及双抗酶联免疫吸附法 (EUA)分别进行FIB、vWF的测定 ,并测定血压、糖化血红蛋白等指标。结果 :糖尿病患者血浆vWF、FIB水平较正常对照组明显升高 (P <0 .0 1) ,合并糖尿病视网膜病变组较无并发症组显著升高 (P <0 .0 1)。结论 :血浆FIB及vWF水平的升高与糖尿病视网膜病变的发生发展有关。     

心室辅助装置引发血管性血友病因子损伤的检测方法研究

目的由于血液成分血管性血友病因子(von Willebrand factor,vWF)的机械损伤现象发现和研究较晚,至今仍然定义模糊且缺乏相关的评价标准,非常不利于心室辅助装置(ventricular assist device,VAD)的创新设计与发展。本文通过研究分析,提出检测vWF受高剪切应力损伤的实验方法。方法人血作为基础血样,离心式血泵BPX-80剪切前后的猪血作为测试血样,通过免疫印迹法,将电泳分离的vWF转移到膜上,然后用特异性抗体检测膜上vWF多聚体的分子量分布情况,根据vWF分子量的灰度值比值,定量分析vWF多聚体的机械损伤,整个实验过程包括制胶、电泳、转印、免疫反应和显色5部分。结果得到条带清晰完整且容易区分的vWF分子量分布图。其中健康人血vWF高分子量、中分子量、低分子量区域的灰度值比值与文献中健康人血vWF的实际比值相一致。BPX-80血泵剪切猪血中高分子量vWF与未经剪切的猪血高分子量vWF灰度值的比值随时间呈下降趋势,表明BPX-80血泵剪切对猪血中vWF的降解情况。结论成功设计并建立了vWF多聚体分析实验方法,为此后制定vWF机械损伤的标准化体外评价方案提供重要参考。     

2A型血管性血友病vWF的基因突变

目的 研究中国人２Ａ型血管性血友病（ｖｏｎ Ｗｉｌｌｅｂｒａｎｄ ｄｉｓｅａｓｅ,ｖＷＤ）的分子病理机理,了解 ｖＷＤ的临床表现型和基因型的相关性。方法 对１２６例先天性出血性疾病患者进行了出血时间、ｖＷＦ：Ａｇ、ＦⅧ：ＣＡｇ、瑞斯脱素诱导的血小板聚集率的检测和ｖＷＦ的血浆多聚物分析。对２Ａ型血管性血友病的患者用多聚酶链反应、变性梯度凝胶电泳,结合测序的方法进行了基因突变的研究。结果 确诊２Ａ型血     

人vWF-A1区蛋白的表达及其对血小板聚集的抑制作用

目的:进一步研究血栓形成的机制,开发抗血栓药物。方法: 应用基因重组技术在大肠杆菌中表达人vWF-A1区蛋白,经过纯化、复性,获得重组蛋白(rvWF-A1),同时用流式细胞术检测rvWF-A1与血小板膜糖蛋白血小板膜糖蛋白(glycoprotein, GP)Ib的结合能力,应用血小板聚集仪测定rvWF-A1对瑞斯托霉素(ristocetin)诱导的血小板聚集抑制作用。结果: 重组表达载体pQE-31-vWF-A1在大肠杆菌M15中得到高效表达,表达的重组蛋白量占菌体总蛋白的30%,Ni-NTA agrose柱纯化后,其纯度为95%,经复性的rvWF-A1蛋白具有良好的生物学活性。它可与血小板模糖蛋白血小板膜糖蛋白GPIb结合,阳性率为78.6%;它可以抑制ristocetin诱导的血小板聚集,抑制率为84.7%。结论: 在原核细胞中可以成功地高效表达人vWF-A1区蛋白,该重组蛋白有可能开发为有效的抗血栓药物。     

博莱霉素肺纤维化小鼠血管内皮细胞的研究

目的:研究C57BL/6小鼠肺微小血管内皮细胞血栓调节蛋白(TM)与因子Ⅷ相关抗原(vWf)的分布及博莱霉素(BLM)致肺纤维化过程中,血管内皮细胞亚型的转变。方法:采用双重免疫荧光染色及荧光强度定量分析法。结果:①正常C57BL/6小鼠肺泡毛细血管内皮细胞表面显示多数连续性线样TM荧光而vWf较少阳性,肺微小血管内皮细胞呈现vWf阳性。②BLM组小鼠内皮细胞TM荧光明显弱于正常,而vWf荧光显著强于正常水平。结论:①正常C57BL/6小鼠肺泡毛细血管内皮细胞以TM表达为主型,而非vWf表达为主型。②BLM致肺纤维化过程中,肺血管内皮细胞由以TM表达为主型转变为以vWf表达为主型,两抗原可被认为是内皮细胞损伤的标志物。     

Intense exercise increases shear-induced platelet aggregation in men through enhancement of von Willbrand factor binding, glycoprotein IIb/IIIa activation, and P-selectin expression on platelets

Vigorous exercise transiently enhances the risk of primary cardiac arrest. Shear-induced platelet aggregation (SIPA) is an important mechanism in arterial thrombogenesis. This study investigates whether intense exercise affects SIPA, and elucidates mechanisms that underlie SIPA. Eighteen sedentary healthy men engaged in intense exercise (about 80% of maximal oxygen consumption) for 40 min on a bicycle ergometer. Platelet aggregation, binding of von Willebrand factor (vWF) to platelets, and activation of glycoprotein (GP) IIb/IIIa and expression of P-selectin on platelets induced by shear stress were analyzed both before and immediately after exercise. Analytical results demonstrated that: (1) the levels of plasma vWF antigen and activity were enhanced after intense exercise, (2) intense exercise increased either shear- or ristocetin-induced platelet aggregation and was accompanied by an increase in vWF binding to platelets and vWF-mediated GP IIb/IIIa activation at high shear flow, and (3) shear-induced P-selectin expression in the absence or the presence of exogenous vWF was enhanced by intense exercise. Therefore, we conclude that intense exercise promotes the extent of SIPA, possibly by enhancing the ability of vWF to bind to platelets and the subsequent activation of GP IIb/IIIa complexes, as well as the expression of P-selectin in response to shear stress, which in turn may augment the risk of vascular thrombosis.     

Direct demonstration of radiolabeled von willebrand factor binding to platelet glycoprotein Ib and IIb-IIIa in the presence of shear stress

In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were:¹²⁵I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and³⁵S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300–360×10³/μl) of radiolabeled vWF was maximum at 60–120 dynes/cm² evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.     

肾综合征出血热患者肾脏超声、血管性血友病因子和肝肾功能的变化及其临床意义

目的 研究肾综合征出血热(HFRS)患者肾脏彩色多普勒超声、血浆血管性血友病因子(vWF)、肝肾功能的变化及临床意义.方法 16例HFRS患者按病情分为轻、重两组,每组各8例,按病期进行肾脏二维及彩色多普勒超声检测,同时采血用酶联免疫吸附试验(ELISA)检测vWF,用自动生化仪检测血尿素氮(BUN)、肌酐(Cr)、丙氨酸氨基转移酶(ALT),用自动血细胞分析仪检测血小板参数;另取30名健康者作对照.结果 HFRS急性期左肾体积、段动脉(SRA)阻力指数(RI)及血浆vWF均显著升高,ALT、BUN也显著上升,在发热期轻、重二组相比有统计学差异(左肾体积:325.12 ±28.53v368.82 ±24.83,P＜0.05;左肾段动脉RI:0.59±0.02 v 0.66±0.03,P＜0.05;vWF:311.55 ±99.98μg/L v 356.36±54.21μg/L,P＜0.05)与BUN、ALT的变化基本一致,但血小板的变化则正相反.结论 HFRS患者肾损害各期的声像图与其临床病理变化密切相关,超声能动态观察病情各期肾损害的轻重,并能估计HFRS病情轻重,判断HFRS患者的临床治疗效果及其预后.肾脏超声检查具有准确、迅速、方便及无痛苦的优点.