A1500E突变对血管性血友病因子的ADAMTS13依赖性水解的影响

目的 研究血管性血友病因子(VWF) A1500E突变体对金属蛋白酶ADAMTS13敏感性的改变,为VWF-A1500E突变导致2A型血管性血友病(VWD)的发病机制提供直接依据.方法 将野生型VWF质粒和A1500E突变体VWF质粒分别瞬时转染HeLa细胞,收集并浓缩培养上清,分别用重组人ADAMTS13( rADAMTS13)进行水解,然后通过十二烷基硫酸钠-琼脂糖凝胶电泳进行VWF多聚体分析,观察与野生型VWF相比,A1500E突变体VWF对ADAMTS13的敏感性有无改变.结果 体外表达研究结果显示WT-VWF和A1500E突变体VWF的表达上清中VWF:Ag的平均含量分别为1.10 U/ml和0.78 U/ml,突变体细胞裂解液中的VWF:Ag表达量为野生型的90.6％,两者差异均无统计学意义(P＞0.05).VWF多聚体电泳显示突变体VWF与WT-VWF的多聚体分布亦无明显差别.在无尿素和盐酸胍等变性剂的静态条件下,rADAMTS13即可对A1500E突变体VWF进行有效地水解,VWF多聚体分析显示大中分子量VWF多聚体明显减少和小分子量VWF多聚体明显增多;相反,野生型VWF在非变性条件下则无被rADAMTS13水解的证据.结论 A1500E突变导致突变体VWF对ADAMTS13的敏感性异常性增高,符合第二组2A型VWD的突变特点.     

无流体剪切力条件下ADAMTS13裂解内皮细胞上特大血管性血友病因子的研究

目的：研究血管性血友病因子裂解酶（ADAMTS13）在无流体剪切力下裂解内皮细胞上特大血管性血友病因子（ULVWF）的分子机制,为探明血栓性血小板减少性紫癜（TTP）和其他血栓性疾病的发病机理提供理论依据。方法：通过免疫荧光显微镜观察ADAMTS13在无流体剪切力下裂解内皮细胞表面上ULVWF的情况,采用ELISA测定不同条件下培养基中VWF抗原量的变化。ELISA和Western blot分别测定有无流体剪切力或凝血因子VIII(FVIII)条件培养基中的VWF和蛋白水解片段的数量。多聚体分析评估ADAMTS13裂解内皮细胞上ULVWF的情况。将组胺刺激的人脐静脉内皮细胞（HUVEC）与ADAMTS13和各种N-和C-末端截断的突变体一起孵育,通过免疫荧光显微镜观察与细胞保持结合的ULVWF,ELISA测定内皮细胞释放出的ULVWF,确定降解内皮细胞上ULVWF所需的ADAMTS13结构域。结果：在无流体剪切力下,重组ADAMTS13和血浆ADAMTS13迅速降解了内皮细胞表面上新形成的ULVWF。ULVWF的蛋白水解过程依赖于培养时间、ADAMTS13浓度和剪切力。ADAMTS13...     

新生儿窒息后早期血浆血管性假血友病因子水平测定的临床意义

目的 探讨新生儿窒息后早期血浆血管性假血友病因子(VWF)的水平变化规律及其临床意义.方法 检测45例不同程度窒息新生儿的血浆VWF水平,同时测定血小板,并与30例正常新生儿比较.结果 新生儿窒息,其血浆VWF水平有不同程度的增高,轻度窒息组和重度窒息组血浆VWF水平明显高于对照组,差异有统计学意义(P<0.01);且重度窒息组血浆VWF水平变化明显高于轻度窒息组(P<0.05).正常对照组与轻度窒息组比较,血小板变化不明显(P>0.05),重度窒息组血小板稍下降.结论 新生儿窒息后早期存在内皮细胞损伤及凝血功能异常,血浆VWF水平异常程度与窒息程度有关,测定血浆VWF水平是反映血液高凝状态,估计疾病严重程度的一个指标,同时对临床选用抗凝药物治疗及疗效观察、预后判断,均有一定的指导意义.     

ADAMTS13依赖性血管性血友病因子水解相关影响因素的研究进展

血浆中血管性血友病因子(vWF)多聚体的相对分子质量的大小主要是由血浆中的血管性血友病因子裂解酶(ADAMTS13)通过剪切vWF A2功能域内的Tyr1605-Met1606之间的肽键而实现的.ADAMTS13对vWF的水解是受多种因素调控或影响的,此种ADAMTS13依赖性vWF水解过程的改变具有重要的生理或病理意义.     

血清血管性血友病因子在不同中医证型脓毒症中的变化

目的 探讨脓毒症内皮细胞功能在中医证型动态整体观察中的意义.方法 将68例脓毒症住院患者按中医卫气营血辨证方法分为气分证组(23例)、营分证组(28例)、血分证组(17例);以26例非脓毒症患者作为对照组.分别取各组患者空腹股静脉血,用放射免疫法检测血清血管性血友病因子抗原(vWf:Ag)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IL-2、IL-4和白细胞计数(WBC).结果 除IL-2外,脓毒症组所有参数值均显著高于对照组(P均<0.05).血分证组vWf:Ag水平明显高于营分证组[(178.6±51.8)%比(129.1±28.7)%,P<0.05].脓毒症各组间,营分证组IL-4、WBC均高于气分证组,血分证组WBC高于营分证组(P均<0.05),而TNF-α、IL-6、IL-2各脓毒症组间比较差异无统计学意义.结论 脓毒症vWf水平随着中医证型的变化而变化;动态观察vWf水平可作为脓毒症辨证的相关参考指标.     

不同类型冠心病患者血管性假血友病因子、氧化型低密度脂蛋白抗体的检测

目的探讨血管性假血友病因子（VWF）、氧化型低密度脂蛋白抗体（oxLDL-Ab）与冠心病严重程度及斑块稳定性的关系。方法根据临床诊断和冠状动脉造影结果，分别测定22例急性心肌梗死（AMI）、22倒不稳定型心绞痛（UAP）、21例稳定型心绞痛（SAP）患者和26例健康对照者血清oxLDL-Ab和血浆VWF的含量并进行比较，同时评估oxLDL-Ab与VWF之间的相关性。结果AMI组和UAP组oxLDL-Ab和VWF的含量明显高于SAP组和正常对照组（均为P〈0．05），Logistic回归分析发现血清oxLDL-Ab与血浆中VWF含量呈正相关（r=0．76，P〈0．01）。ACS组中三支血管病变组oxLDL-Ab与VWF水平明显高于单支病变组和正常对照组。结论oxLDL-Ab和VWF的含量变化不仅反映冠心病患者病变的严重程度。而且可以评价冠心病斑块的稳定性。     

脓毒症合并弥散性血管内凝血患者血清G-CSF水平及意义

目的 检测脓毒症及合并弥散性血管内凝血(DIC)患者血清中粒细胞集落刺激因子(G-CSF)水平,并判断其对脓毒症患者发展成为DIC的诊断意义.方法 严重脓毒症患者28例(SS组),脓毒症合并DIC患者12例(SSD组),同期门诊体检者20例为对照组,收集临床及实验室参数,计算APACHEⅡ评分和DIC评分;采用酶联免疫吸附测定(ELISA)方法检测血清G-CSF水平.结果 SS组和SSD组患者血G-CSF水平较对照组明显升高(P＜0.05),但SS组和SSD组患者之间血清G-CSF水平差异无统计学意义(P＞0.05).相关分析显示,G-CSF与WBC、PCT、APACHEⅡ评分呈显著正相关.ROC曲线图显示,血清G-CSF的AUC =0.887,95％CI为(0.825,0964) (P ＜0.05).结论 血清G-CSF水平主要与脓毒症严重程度有关,与DIC的发病机制无明显相关性,可作为脓毒症严重程度判断的实验室证据.     

急性心肌梗死和脑梗死患者血浆中ADAMTS13的测定及意义

目的 研究急性心肌梗死(AMI)及急性脑梗死(AIS)患者血浆中血管性血友病因子(vWF)裂解酶(ADAMTS13)抗原和活性变化情况,探讨ADAMTS13在动脉血栓性疾病患者发病中的作用及意义.方法 用Frests-WF73试剂盒检测血浆中ADAMTS13的活性;用ELISA法检测ADAMTS13抗原含量;并对患者血浆vWF多聚物进行电泳分析.结果 正常对照组、AMI组和AIS组ADAMTS13抗原含量分别为(878±198)、(618±188)和(702±155)U/L;活性分别为(81.7±13.9)%,(59.2±22.1)%和(65.4±15.8)%.AMI组和AIS组ADAMTS13抗原含量及活性均显著低于正常对照组(P《0.01);vWF多聚物分析未见异常.结论 AMI及AIS患者血浆ADMATS13抗原及活性均降低,提示ADAMTS13的减少与动脉血栓性疾病的发病相关.     

血管性血友病因子裂解蛋白酶活性的检测方法及评估

血管性血友病因子裂解酶(ADAMTS13,vWF-CP)裂解血浆中具有高黏附能力的超大分子量血管性血友病因子(UL-vWF),防止血小板因其引起聚集形成血栓。ADAMTS13活性异常是临床上血栓性血小板减少性紫癜(TTP),特别是遗传性TTP和特发性TTP发病的基础。其活性的检测在TTP临床诊断和治疗上具有日益重要的意义。目前报道的一些用于检测ADAMTS13活性的方法有十余种,本文对此进行综述。     

椎动脉型颈椎病患者血管性假血友病因子检测的临床意义

目的探讨血管性假血友病因子（VWF）检测在椎动脉型颈椎病中的临床意义。方法选取109例椎动脉型颈椎病患者（椎动脉型组）、30例神经根型颈椎病患者（神经根型组）和30例正常者（正常对照组）。采用酶联免疫吸附双抗体夹心法（ELISA）测定血浆VwF含量，同时对椎动脉型组中30例患者作颅脑CT检查分析。结果椎动脉型组患者血浆VWF含量显著高于神经根型组（P〈0．01），亦显著高于正常对照组（P〈0．01）。而CT检查发现，随着病程延长，脑梗死的检出率明显升高。结论血浆VWF浓度升高可作为椎动脉型颈椎病的辅助诊断指标之一。在椎动脉型颈椎病患者中，血浆VWF浓度升高提示有发生脑梗死的危险。     

哮喘患者白细胞介素13与临床特征、糖皮质激素疗效相关性研究

目的:探讨白介素13(IL-13)对支气管哮喘患者临床特征和糖皮质激素治疗的影响.方法:采用ELIAS分别检测34例发作期哮喘患者(其中14例激素敏感型和10例激素抵抗型口服强的松治疗1周)、18例缓解期患者和30例健康对照者血浆IL-13浓度.结果:52例哮喘患者和30例健康人血浆IL-13质量浓度(65.65±3.38 vs 25.83±3.59)比较,差异有显著性(P＜0.05).发作期(34例)和缓解期(18例)哮喘患者血浆IL-13质量浓度(72.03±3.72 vs 48.67±4.22)比较,差异有显著性(P＜0.05).在34例发作期哮喘患者中,轻、中、重度者其血浆IL-13质量浓度(52.35±2.98 vs 65.12±2.56 vs 76.57±2.36)相互间比较,差异有显著性(P＜0.05).激素敏感型哮喘经强的松治疗1周后血浆IL-13质量浓度较治疗前明显下降(73.03±3.72 vs 55.67±4.22),差异有显著性(P＜0.05);激素抵抗或依赖型哮喘经强的松治疗1周前后血浆IL-13质量浓度比较(70.35±2.98 vs 68.57±2.36),差异无显著性(P＞0.05).结论:血浆IL-13参与哮喘的病理生理过程,可作为判断哮喘患者病变严重程度的指标之一.IL-13参与调节糖皮质激素作用机制.     

Validation of the PLASMIC score at a University Medical Center

Background

The PLASMIC score was recently described as a convenient tool for predicting ADAMTS13 activity ≤10% in patients with possible thrombotic thrombocytopenic purpura (TTP), while awaiting the results of this send-out test. The purpose of this study was to validate the PLASMIC score at our University Medical Center.

Elevated procalcitonin and C‐reactive protein as potential biomarkers of sepsis in a subpopulation of thrombotic microangiopathy patients

Thrombotic microangiopathy (TMA) comprises a group of microvascular thrombosis syndromes associated with multiple pathogenic factors. Deficient activity of ADAMTS13 is a pathogenic factor in a subset of TMA patients that provides a strong rationale for plasma exchange treatment. However, the subset of TMA patients with normal ADAMTS13 activity remains a heterogeneous group of patients in which the appropriate treatment is not well understood. In addition to the common forms of TMA thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome, the differential diagnosis of TMA may include sepsis, autoimmune disorders, and disseminated intravascular coagulation. Optimal treatment of TMA depends on timely recognition of treatable pathogenic factors. We hypothesized that sepsis is a rapidly identifiable pathogenic factor in a subset of TMA patients. To test this hypothesis, we retrospectively measured the rapid biomarkers of sepsis C‐reactive protein (CRP) and procalcitonin (PCT), in a repository of pretreatment plasma samples from 61 TMA patients treated with plasma exchange. Levels were analyzed in 31 severely ADAMTS13‐deficient and 30 ADAMTS13‐normal patients. None of the 31 patients with severe deficiency of ADAMTS13 had elevated PCT. However, 11 of 30 (37%) non‐ADAMTS13‐deficient patient samples were strongly positive for PCT. These patient samples also had a >10‐fold higher median CRP level than patients with normal PCT. We conclude that rapid assays may help identify sepsis in a subset of TMA patients. J. Clin. Apheresis, 2009. © 2009 Wiley‐Liss, Inc.     

Assessment of real‐time myocardial uptake and enzymatic conversion of hyperpolarized [1‐13C]pyruvate in pigs using slice selective magnetic resonance spectroscopy

Hyperpolarization of ¹³C‐labeled energy substrates enables the noninvasive detection and mapping of metabolic activity, in vivo, with magnetic resonance spectroscopy (MRS). Therefore, hyperpolarization and ¹³C MRS can potentially become a powerful tool to study the physiology of organs such as the heart, through the quantification of kinetic patterns under both normal and pathological conditions. In this study we assessed myocardial uptake and metabolism of hyperpolarized [1‐¹³ C]pyruvate in anesthetized pigs. Pyruvate metabolism was studied at baseline and during dobutamine‐induced stimulation. We applied a numerical approach for spectral analysis and kinetic fitting (LSFIT/KIMOfit), making a comparison with a well‐known jMRUI/AMARES analysis and γ‐variate function, and we estimated the apparent conversion rate of hyperpolarized [1‐¹³ C]pyruvate into its downstream metabolites [1‐¹³C]lactate, [1‐¹³C]alanine and [¹³C]bicarbonate in a 3 T MR scanner. We detected an increase in the apparent kinetic constants (k_PX) for bicarbonate and lactate of two‐fold during dobutamine infusion. These data correlate with the double product (rate‐pressure product), an indirect parameter of cardiac oxygen consumption: we observed an increase in value by 46 ± 11% during inotropic stress. The proposed approach might be applied to future studies in models of cardiac disease and/or for the assessment of the pharmacokinetic properties of suitable ¹³C‐enriched tracers for MRS. Copyright © 2012 John Wiley & Sons, Ltd.     

Suppression of phosphorylation of extracellular-signal-regulated kinase and p38 mitogen-activated protein kinase in polymorphonuclear leukocytes by the proton pump inhibitor lansoprazole

Lansoprazole (LPZ) is a proton pump inhibitor that suppresses gastric secretion and exerts anti-inflammatory effects on immune cells. Recently, LPZ has been used for the treatment of peptic ulcer and gastritis, which can be caused by Helicobacter pylori, due to its potent acid-suppressive effects. We focused the aim to the anti-inflammatory effects on the over-activation of neutrophils, and investigated the effects of LPZ on the signal transduction of the mitogen-activated protein kinase (MAPK) family. LPZ slightly phosphorylated p38 MAPK of neutrophils at a concentration of 10 μg/ml, but did not phosphorylate extracellular-signal regulated kinase (ERK) 1/2. Pretreatment of neutrophils with (1–5 μg/ml) LPZ strongly attenuated the phorbol-12-myristate-13-acetate-stimulated phosphorylation of ERK1/2, and LPZ slightly suppressed the lipopolysaccharide (LPS)- and N-formylmethionylleucylphenylalanine-stimulated phosphorylation of p38. ERK1/2 produces the mitochondrial anti-apoptotic proteins, and the signaling pathway from LPS and N-formylmethionylleucylphenylalanine to p38 is the main pathway for reactive oxygen species production. The mechanism of anti-inflammatory effect of LPZ on hyper-activated neutrophils is suggested to be the suppression of signal transduction of ERK1/2 and p38 MAPK.     

Microangiopathic hemolytic anemia in pregnancy

Microangipathic hemolytic anemia (MAHA) is a serious diagnosis and difficult to manage in pregnant patients as multiple life threatening conditions could present with MAHA. ADAMTS13 enzyme activity can be affected during pregnancy with multiple factors. A persistent extremely low ADAMTS13 enzyme activity levels, without an inhibitor, after the delivery was an important factor to establish the diagnosis. We present a case of likely congenital ADAMST13 deficiency that manifested for the first time in a pregnant woman at week 37 of pregnancy.     

罕见的ins(22;9)t(9;13)/Ph~-慢性髓系白血病实验研究

本文报告1例罕见的具有ins(22;9)t(9;13)的Ph(-)慢性髓系白血病。骨髓细胞经24小时短期培养法制备染色体标本,采用G显带技术进行核型分析;用9号、22号全染色体涂染探针进行染色体涂染;用LSI bcr/abl双色双融合探针进行FISH分析;用实时荧光定量PCR法检测bcr/abl融合基因转录本及其拷贝数。结果表明,染色体核型为45,XX,der(9)t(9;13)(q34;q10),-13[20],abl基因插入到der(22)形成插入性的bcr/abl基因重排;实时荧光定量PCR检测到bcr/abl融合基因。结论:插入性的bcr/abl基因重排是t(9;22)的一种罕见的变异类型,可用分子学方法检测,但全染色体涂染及常规的染色体显带分析容易导致误诊。     

APRIL／BlyS表达与原发性胆汁性肝硬化的关联性研究

目的 探讨APRIL/BlyS在原发性胆汁性肝硬化患者外周血单个核细胞中的mRNA表达水平以及与体液免疫水平的关联性.方法 设计特异性的引物和探针,以人基因GAPDH为内参照,用实时定量PCR的方法检测42例健康人和56例原发性胆汁性肝硬化患者外周血单个核细胞中APRIL,BlyS的mRNA表达水平,采用ELISA方法在全自动酶标仪上检测血清中IgG,IgA,IgM的浓度.结果 与健康人比较,原发性胆汁性肝硬化患者外周血中APRIL,BlyS mRNA的表达升高(P<0.01),患者血清中IgM显著增高(P<0.01),患者中BlyS mRNA表达与IgM成显著正相关(r=0.763,P<0.01),而APRIL mRNA的表达与IgM的水平无相关性.结论 ARPIL,BlyS系统及其受体可能参与原发性胆汁性肝硬化的疾病进程,为监控原发性胆汁性肝硬化的病情和有效治疗提供了新的依据.     

伴t(1;19)(q23;p13)和E2A-PBX1成人急性T淋巴细胞白血病一例报告附文献复习

目的 总结1例伴有t(1;19)和E2A-PBX1融合基因阳性的成人T细胞急性淋巴细胞白血病(ALL)患者的诊疗体会.方法 对1例成人T细胞ALL患者进行染色体核型分析,流式细胞术检测免疫表型,同时进行融合基因多重RT-PCR扩增.结果 患者染色体核型为47,XY,9p+,15p+,17q-,der(19),t(1;19)(q23;p13)[5]/46,XY[15].E2A-PBX1融合基因阳性表达.给予hyperCVAD(环磷酰胺+长春新碱+阿霉素+地塞米松)方案治疗后患者获血液学完全缓解,染色体核型复查为46,XY[10],E2A-PBX1融合基因检测阴性.结论 E2A-PBX1+ t(1;19)也可以发生于T细胞ALL,E2a-PBX1白血病细胞在不同的癌基因协同信号或微环境下转化方向可变.     

Real‐time cardiac metabolism assessed with hyperpolarized [1‐13C]acetate in a large‐animal model

Dissolution‐dynamic nuclear polarization (dissolution‐DNP) for magnetic resonance (MR) spectroscopic imaging has recently emerged as a novel technique for noninvasive studies of the metabolic fate of biomolecules in vivo. Since acetate is the most abundant extra‐ and intracellular short‐chain fatty acid, we focused on [1‐¹³C]acetate as a promising candidate for a chemical probe to study the myocardial metabolism of a beating heart. The dissolution‐DNP procedure of Na[1‐¹³C]acetate for in vivo cardiac applications with a 3 T MR scanner was optimized in pigs during bolus injection of doses of up to 3 mmol. The Na[1‐¹³C]acetate formulation was characterized by a liquid‐state polarization of 14.2% and a T_1Eff in vivo of 17.6 ± 1.7 s. In vivo Na[1‐¹³C]acetate kinetics displayed a bimodal shape: [1‐¹³C]acetyl carnitine (AcC) was detected in a slice covering the cardiac volume, and the signal of ¹³C‐acetate and ¹³C‐AcC was modeled using the total area under the curve (AUC) for kinetic analysis. A good correlation was found between the ratio AUC(AcC)/AUC(acetate) and the apparent kinetic constant of metabolic conversion, from [1‐¹³C]acetate to [1‐¹³C]AcC (k_AcC), divided by the AcC longitudinal relaxation rate (r₁). Our study proved the feasibility and the limitations of administration of large doses of hyperpolarized [1‐¹³C]acetate to study the myocardial conversion of [1‐¹³C]acetate in [1‐¹³C]acetyl‐carnitine generated by acetyltransferase in healthy pigs. Copyright © 2014 John Wiley & Sons, Ltd.