连续性血液净化治疗对热射病患者组织因子的影响

目的：研究连续性血液净化（CBP）治疗对热射病患者组织因子的影响。方法：以34例热射病导致的多器官功能不全综合征（MODS）患者作为研究对象,所有患者行持续静脉血液滤过（CVVH）48h;以健康成人为对照。在治疗前以及CBP治疗各时间点,以ELISA法测定患者血清组织因子（TF）、可溶性血栓调节蛋白（sTM）、可溶性E选择素（sE-selectin）的水平,以流式细胞术测定单核细胞膜表面TF活性。用Pearson积差相关系数,研究血清TF与单核细胞膜表面TF活性、血清sTM以及sE-selectin的相关性。结果：与正常对照组比较,热射病患者血清TF、sTM和sE-selectin水平以及单核细胞膜表面TF活性明显增高（P〈0.01）。与治疗前比较,CBP治疗导致血清TF、sTM和sE-selectin水平、单核细胞膜表面TF活性明显下降（P〈0.05）。血清TF与单核细胞膜表面TF活性、血清sTM以及sE-selectin有显著相关性（P〈0.05）。结论：CBP可以通过抑制单核细胞和内皮细胞产生TF以及直接清除血清TF,达到降低血清中过度产生的TF作用。     

急性冠脉综合征患者血小板和白细胞异常表达组织因子的研究

本研究观察急性冠脉综合征（ACS）患者,稳定性心绞痛（SA）患者及健康对照循环中血小板、血小板白细胞聚集体（PLA）和血小板单核细胞聚集体（PMP）组织因子（TF）的表达情况,并探讨其在ACS发病机制中的作用。收集26例ACS患者,29例SA患者及25名正常人（作为对照组）,外周血标本,分离获得单核细胞及富含血小板血浆,用RT-PCR法分别检测组间单核细胞及血小板TF-mRNA的表达,通过流式细胞仪检测组间血小板、PLA和PMP表达TF的比例。结果表明,ACS组、SA组、正常对照组血小板TF mRNA平均表达水平分别为3.11±0.51、1.88±0.78和0.7±0.10,ACS组单核细胞、血小板TF mRNA表达水平与正常对照组有差异（分别为P=0.03,P=0.05）。ACS组患者血小板表达TF比例明显高于SA组及正常对照组（P=0.02）,3组的TF阳性的PLA百分比（%UR）分别为2.7±0.6、0.8±0.2及0.5±0.1（P=0.001）。ACS组TF阳性的血小板单核细胞百分比（%UR）为2.5±0.6,显著高于SA组的1.2±0.3（P=0.02）及对照组的0.8±0.2（P=0.04）。结论：ACS患者血小板及白细胞高表达TF,促进了血小板的活化、凝血和血栓形成,进一步促进高凝状态。TF促血栓形成和促局部炎症作用,协同血小板和白细胞共同参与ACS的发病,最终与ACS疾病发生发展密切相关。     

急性脑梗死患者外周血血小板表面P-选择素阳性表达率变化及药物干预影响

目的 探讨血小板活化因子P-选择素在急性脑血管疾病患者中的改变及应用奥扎格雷钠治疗后的变化。方法采用流式细胞仪测定急性脑梗死患者及正常对照组外周血血小板表面P-选择素阳性表达情况，对比观察治疗前后P-选择素的变化。结果急性脑梗死患者外周血血小板表面P-选择素阳性表达率显著高于正常对照组（P〈0．01）；应用奥扎格雷钠治疗后的外周血血小板表面P-选择素阳性表达率明显低于正常对照组（P〈0．01）。结论血小板活化在急性脑梗死的发生、发展中起重要作用；P-选择素可作为选择药物治疗的一项可靠指标，奥扎格雷钠可减少血小板活化。     

血小板胶原受体糖蛋白Ⅵ在脑梗塞中的临床意义

目的探讨脑梗塞患者外周血血小板胶原受体糖蛋白VI和CD62P含量以及临床意义。方法用流式细胞仪检测脑梗塞患者活化血小板标志物GPVI和CD62P．并与对照组比较。结果脑梗塞患者血小板表面表达GPVI和CD62P的荧光强度分别为20．52±5．80和31．52±15．67,均明显高于正常对照组（P〈0．01）。结论脑梗塞患者血栓的形成与血小板的活化密切相关,GPVI和CD62P的检测可以作为脑梗塞早期诊断的一项较特异指标。     

急性冠脉综合征妊娠相关血浆蛋白-A与肿瘤坏死因子-α相关性的研究

【目的】探讨急性冠脉综合征（ACS）患者妊娠相关血浆蛋白A（PAPP—A）血清水平和外周血单核细胞表达的变化及其与肿瘤坏死因子-α（TNF-α）的相关性。【方法】测定18例不稳定性心绞痛（UAP组）、37例急性心肌梗死（AMI组）和15例稳定性心绞痛（SAP组）及15例正常对照者的血清PAPP-A、TNF-α水平。同时分离外周血单核细胞，应用RT-PCR检测PAPP-A和TNF-α的mRNA表达。【结果】SAP组血清PAPP—A水平和单核细胞PAPP—A的mRNA表达水平与正常对照组相比，无显著差异；而在UAP组和AMI组均显著高于正常对照组（P〈0．01）和SAP组（P〈0．01）。ACS组患者的血清PAPP—A水平与血清TNF-α水平呈显著正相关（r=0．712，P〈0．01），同时外周血单核细胞PAPP-AmRNA表达水平与TNF-αmRNA表达水平也呈显著正相关（r=0．733，P〈0．01）。【结论】PAPP-A可能是由活化的单核细胞合成并分泌到动脉粥样硬化斑块内和血循环中，并与炎症密切相关。     

原发性肾小球肾炎患者外周血血小板P-选择素检测的临床意义

目的研究外周血血小板P-选择素检测在原发性肾小球肾炎（PGN）患者诊断、治疗中的临床意义。方法对34例PGN患者经皮肾穿刺活检获得病理诊断,根据肾组织中肾小球内皮细胞增生及硬化程度分为3组,采用流式细胞术（FCM）检测患者及20名对照组外周血血小板P-选择素阳性表达率。结果PGN患者各组外周血血小板P-选择素表达阳性率均高于对照组,差异有统计学意义（F=65．67,P〈0．01）。其中Ⅱ组表达阳性率较Ⅰ组增高（q=6．49,P〈0．01）,Ⅱ组也较Ⅲ组增高（q=5．19,P〈0．01）。结论P．选择素与肾脏疾病关系密切,检测PGN患者外周血血小板P-选择素为临床抗黏附治疗提供实验依据。     

人外周单个核细胞来源树突细胞成熟与肺炎支原体膜脂蛋白的影响

目的：实验着眼于肺炎支原体膜脂蛋白对人外周血单个核细胞来源的树突细胞的表型（CD83，CD86）及分泌细胞因子的作用，探讨肺炎支原体加重哮喘的机制是否是改变了树突细胞的性状。 方法：①对象：所有外周血采自正常人，志愿献血者为武汉大学医学院2005级硕士博士研究生6人：实验用肺炎支原体膜脂蛋白购自广州华银医药科技有限公司。②实验过程及分组：从肘静脉采集志愿者20mL新鲜全血，以不连续密度梯度离心法及贴壁法获取单核细胞，加入重组人粒细胞-巨噬细胞集落刺激因子，重组人白细胞介素4培养5d得到不成熟树突细胞后，分别予肺炎支原体膜脂蛋白，脂多糖和空白刺激再培养2d。③评估：用流式细胞仪检测各组树突细胞表面表达CD83，CD86的情况，用酶联免疫吸附法检测各组树突细胞分泌白细胞介素12的水平。 结果：①肺炎支原体膜脂蛋白组树突细胞表达CD83，CD86高于未刺激组（P〈0．01，P〈0．01）;脂多糖组树突细胞表达CD83，CD86高于末刺激组（P〈0．01，P〈0．01）;肺炎支原体膜脂蛋白组树突细胞表达CD83较脂多糖组无差异，表达CD86高于脂多糖组（P〈0．05）。②肺炎支原体膜脂蛋白组树突细胞分泌白细胞介素12高于未刺激组（P〈0．01）;脂多糖组树突细胞分泌白细胞介素12高于未刺激组（P〈0．01）；肺炎支原体膜脂蛋白组树突细胞分泌白细胞介素12低于脂多糖组（P〈0．05）。 结论：肺炎支原体膜脂蛋白可刺激未成熟树突细胞分化成熟，但较脂多糖刺激的不同，前者刺激成熟的树突细胞在呈递抗原给T细胞时，可进一步使T细胞向Th2极化，导致Th1／Th2平衡失调，从而加重哮喘。     

SIRS与MODS患者外周血单核细胞环氧化酶与血小板活化因子的关系研究

目的了解环氧化酶（COX）和血小板活化因子（PAF）在全身炎症反应综合征（SIRS）与多器官功能障碍综合征（MODS）患者发病机制中的作用。方法选择28例符合美国胸科医师协会／危重病医学会（ACCP／SCCM）提出的SIRS和MODS诊断标准患者，其中SIRS组13例，MODS组15例，另以与息者年龄、性别相匹配的11名健康体检者作为正常对照组。采用淋巴细胞分离液密度梯度离心法分离外周血单核细胞（PBMCs）；用酶联免疫吸附法（ELISA）检测PBMCs中COX-2含量与血小板活化因子乙酰水解酶（PAF—AH）活性；用逆转录-聚合酶链反应（RT—PCR）法检测PBMCs中COX-2与PAF—AH的mRNA表达。结果MODS组患者PBMCs中COX-2含量、PAF—AH活性及两者的mRNA表达均显著高于正常对照组和SIRS组（P均〈0．05），正常对照组与SIRS组间差异无显著性，死亡患者高于存活息者（P均〈0．05）；3组PBMCs中COX-2含量与PAF—AH活性间呈正相关关系（r=0．329，P〈0．05）。死亡息者的外周血白细胞计数、淋巴细胞计数、氧合指数与存活患者比较差异均无显著性，血糖、血肌酐则明显高于存活息者（P〈0．05和P〈0．01），CO2总含量（TCO2）、动脉血pH值均明显低于存活息者（P均〈0．01）。结论COX-2与PAF—AH参与了MODS的发病过程，而且可作为判断SIRS与MODS预后的参考指标；血糖、肌酐、TCO2、pH可作为判断病情及预后的其他参考指标。     

2型糖尿病患者微血管病变与血小板活化关系的研究

目的：探讨血小板活化在糖尿病及其微血管病变发生、发展中的作用。方法：对糖尿病微血管病变患者按尿清蛋白排泄率（UAER）分为正常清蛋白尿（NA）组、微量清蛋白尿（MA）组、临床清蛋白尿（CP）组三组，分别用流式细胞术、血液聚集仪、全自动血细胞分析仪测定血小板内“一颗粒膜蛋白（CD62p）、溶酶体蛋白（CD63）的表达及血小板聚集功能和平均体积，并与30例正常对照组比较。结果：糖尿病微血管病变患者各组血小板膜糖蛋白CD62p、CD63的表达水平及血小板聚集功能和血小板平均体积均增高，与对照组比较差异有显著性（P〈O．01或P〈0．05）；糖尿病微血管病变患者中，CP组CD62p、CD63的表达水平及血小板聚集功能和血小板平均体积显著高于NA组（P〈0．01）和MA组（P〈0．01），MA组又显著高于NA组（P〈0．01或P〈0．05）。结论：CD62p、CD63的表达及聚集功能和平均体积的测定是反映血小板活化的指标，对判断糖尿病微血管病变的发生、发展及早期诊断和早期预防具有重要意义。     

姜黄素联合顺铂对肺癌治疗的体外实验研究

目的探讨炎症因子CD62p（P-选择素）、CD11b、血小板-单核细胞聚合体（PMA）在恶性血液病不同病期的表达特征及临床意义。方法用流式细胞仪检测77例恶性血液病患者及30例正常人CD62p、CD11b及PMA的表达情况。结果恶性血液病初治时CD62P、CD11b、PMA表达水平高于对照（P〈0．05）；急性白血病（AL）缓解时各因子的表达水平低于初治（P〈0．05），部分缓解（PR）时炎症因子的表达水平高于对照（P〈0．05），CR期与对照组比较差异无显著性（P〉0．05）。结论恶性血液病发病时存在易栓状态；急性白血病各病期CD62P、CD11b、PMA的表达不同，可作为临床疗效观察、预后判断的指标之一。     

The role of platelets in decrypting monocyte tissue factor

Although about 80% of tissue factor (TF) extracellular domain antigen present in lipopolysaccharide (LPS)-stimulated monocytes is available at the cell surface, only 10% to 20% of the total extractable TF activity is expressed on the surface of intact monocytes. Thus, most of the TF activity is latent or encrypted in the cell membrane. When coincubated, leukocytes and platelets generate more TF activity than either cell type alone. We have shown that such platelet-promoted enhancement of LPS-induced TF activity in monocytes in whole blood depends on neutrophil involvement in a P-selectin/CD15 (a leukocyte membrane-bound carbohydrate)-dependent reaction. The effect was even more pronounced when both the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and LPS were present during monocyte stimulation. We currently envisage that decryption is mediated through the secretion of TF-rich particles by monocytes. These particles express CD15 and bind P-selectin exposed on either activated platelets or platelet-derived microparticles. Interactions and fusion events, that typically occur between monocytes and platelets, would facilitate the generation of monocytes/monocyte microparticle and platelets/platelet microparticle hybrids, leading to particles rich in decrypted TF activity. In conclusion, platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favors thrombogenesis.     

急性心肌梗死患者组织因子及组织因子途径激活抑制物水平的变化

目的探讨急性心肌梗死(AMI)患者组织因子(TF)及组织因子途径激活抑制物(TF-PI)的变化及临床意义。方法用ELISA法分别检测50例AMI患者及20例健康对照的外周血TF及TFPI的抗原水平。比较15例支架手术患者冠状窦内及外周血的TFPI水平。比较并发糖尿病和/或高脂血症AMI患者与无并发症者的TFAg及TFPIAg水平。结果AMI患者的TFAg及TFPIAg较正常人均明显升高(P<0.01);支架患者冠状窦内的TFPI水平明显低于其外周血(P<0.01);有并发症患者的TF及TFPI水平均明显高于无并发症患者(分别为P<0.01和P<0.05)。结论AMI患者外周血凝血活性增高,并发糖尿病和/或高脂血症AMI患者的凝血活性较无并发症患者高,血栓形成使冠状动脉腔内的TFPI消耗而降低。     

Role of platelets in tissue factor expression by monocytes in normal and hypercholesterolemic subjects. In vitro effect of cerivastatin

Thrombosis is a complication of atherosclerosis and monocytes play a determinant role either in the progression of atherosclerotic plaque or in blood coagulation by way of tissue factor expression. Platelets play a direct role in thrombosis and a hyperfunctional state has been described in hypercholesterolemic subjects. Moreover, platelets seem to be able to enhance monocyte activity. Cholesterol-lowering molecules (statins) are reported to reduce cardiovascular risk, either by decreasing the circulating level of cholesterol or by non-lipidic actions such as the reduction of monocyte and platelet activity. The aim of our study was to investigate the influence of platelets on the expression of tissue factor by monocytes and the effect induced by cerivastatin. We measured tissue factor levels by ELISA and the procoagulant activity of stimulated monocytes by a clotting assay on cellular preparations and whole blood in 40 hypercholesterolemic subjects (22 male, 18 female, mean age 52.7 +/- 12 years, total cholesterol 251.6 +/- 19.9 mg/dl) before and after cerivastatin addition. Tissue factor expression was enhanced in hypercholesterolemic subjects compared with normal subjects (31.6 +/- 7.6 vs. 23 +/- 5.8 pg/cells, P < 0.01). The presence of platelets increased the amount of tissue factor (55.3 +/- 7.3 pg/cells, P < 0.001) and cerivastatin reduced the expression of tissue factor in isolated monocytes, in the mixed cellular system, and in whole blood (19.6 +/- 4.1 pg/cells, P < 0.001). In conclusion, tissue factor expression by monocytes is enhanced in hypercholesterolemic subjects compared with normal controls. Platelets enhance monocyte production of tissue factor, and cerivastatin is able to counteract this prothrombotic mechanism.     

Superantigens from Staphylococcus aureus induce procoagulant activity and monocyte tissue factor expression in whole blood and mononuclear cells via IL-1β

Summary.  Background :  Staphylococcus aureus is one of the most common bacteria in human sepsis, a condition in which the activation of blood coagulation plays a critical pathophysiological role. During severe sepsis and septic shock microthrombi and multiorgan dysfunction are observed as a result of bacterial interference with the host defense and coagulation systems. Objectives : In the present study, staphylococcal superantigens were tested for their ability to induce procoagulant activity and tissue factor (TF) expression in human whole blood and in peripheral blood mononuclear cells. Methods and results : Determination of clotting time showed that enterotoxin A, B and toxic shock syndrome toxin 1 from S. aureus induce procoagulant activity in whole blood and in mononuclear cells. The procoagulant activity was dependent on the expression of TF in monocytes since antibodies to TF inhibited the effect of the toxins and TF was detected on the surface of monocytes by flow cytometry. In the supernatants from staphylococcal toxin-stimulated mononuclear cells, interleukin (IL)-1β was detected by ELISA. Furthermore, the increased procoagulant activity and TF expression in monocytes induced by the staphylococcal toxins were inhibited in the presence of IL-1 receptor antagonist, a natural inhibitor of IL-1β. Conclusions : The present study shows that superantigens from S. aureus activate the extrinsic coagulation pathway by inducing expression of TF in monocytes, and that the expression is mainly triggered by superantigen-induced IL-1β release.     

血小板相关组织因子及其意义的研究

目的探讨正常人血小板是否含有组织因子(TF),血小板相关TF是否具有促凝活性。方法用Sepharose2B凝胶柱分离纯化血小板,ELISA法测定洗涤血小板破碎液TF含量;一期血浆凝固时间测定血小板相关TF促凝活性;RT-PCR法检测正常人血小板相关TF基因的表达。结果正常人血小板破碎液中TF含量为(16.37±6.39)ng/L;胶原活化的血小板可将TF释放到血浆中引起血浆TF水平明显升高(P<0.05);静息血小板无TF的促凝活性,胶原活化的血小板促凝活性显著增高(P<0.01),TF单抗及乏凝血因子Ⅶ(FⅦ)血浆能明显阻断该效应(P<0.01);RT-PCR法在血小板上未能检测到TF mRNA的存在;冠心病患者血小板破碎液TF水平为(20.71±8.78)ng/L,与正常对照相比明显增高(P<0.05),体外未经胶原活化的血小板促凝活性也较正常对照明显增高,并能被TF单抗抑制。结论血小板内含有TF;活化血小板可表现出TF促凝活性;血小板所含的TF可能并非自身合成;冠心病患者血小板相关TF的含量及活性较正常对照明显增高,提示血小板可能可以通过释放TF而参与凝血过程及血栓形成。     

Role of inflammatory mediators in thrombogenesis

The role of inflammation in cardiovascular disease and especially in thrombogenesis has become increasingly recognized as an important component of the overall disease process. Plaque rupture promotes activation of the inflammatory response and increased expression of tissue factor (TF), which in turn acts as one of the major initiators of extrinsic coagulation. It is becoming apparent that the expression of TF on endothelial cells, underlying smooth muscle cells and monocytes is regulated, in part, by proinflammatory cytokines including tumor necrosis factor and IL-1. In addition to initiating coagulation, interaction of TF with the adhesion molecule, P-selectin, has been demonstrated to accelerate the rate and extent of fibrin formation and deposition. P-selectin is expressed on activated platelets and endothelium and serves as the receptor for the endogenous ligand, P-selectin glycoprotein-1 (PSGL-1), expressed on various leukocytic cell types. In addition to mediating transient interactions between endothelial cells and leukocytes, P-selectin has been reported to mediate adherence of platelets to monocytes and neutrophils via specific interaction with PSGL-1. P-selectin is rapidly cleaved off the surface of the platelet membrane and appears in the circulation as a soluble form, which has been reported to be elevated in patients with acute coronary syndromes including unstable angina and non-Q-wave myocardial infarction. This review will focus on the role of cytokines in mediating TF expression and also explore the significance of the relationship between P-selectin and tissue factor in thrombus generation. In addition, possible pharmacological mechanisms to interrupt this disease process will be discussed.     

C-reactive protein contributes to the hypercoagulable state in coronary artery disease

OBJECTIVES: Elevated plasma C-reactive protein (CRP) levels predict coronary events, but it is unclear whether CRP plays a role in thrombosis associated with these events. We investigated tissue factor (TF) induction by CRP on peripheral blood mononuclear cells (PBMC) from patients with coronary disease. PATIENTS AND METHODS: PBMC from 35 patients with stable angina (SA) in study 1, 10 male patients with SA, 10 with unstable angina (UA) and 10 matched controls in study 2, and 25 patients with inflammatory disorders (ID) and 24 normal controls in study 3 were stimulated with CRP, interferon-gamma (IFN) or lipopolysaccharide (LPS), or their combination. PBMC from additional normal donors were also stimulated with CRP in adherent and non-adherent conditions, and TF activity, antigen and mRNA expression detected. RESULTS: CRP (5-25 microg mL(-1)) dose dependently induced more TF on PBMC from SA patients than 42 contemporary controls (P = 0.001, study 1). Compared with controls, patients with SA or UA had higher basal, and much higher CRP- or CRP/LPS-induced monocyte TF activity although serum CRP levels were similar (study 2). IFN induced monocyte TF activity in patients with angina, but not in controls. Basal or CRP-induced TF levels did not differ between controls and ID, even though ID patients had much higher serum CRP levels (study 3). CRP-induced monocyte TF activity correlated with serum CRP levels in controls (P = 0.005) and ID (P = 0.007) in study 3, but not in patients with angina (P =0.84) in study 2. CRP induced more TF activity, protein and mRNA under adherent than non-adherent conditions implying that it may mainly target macrophages in lymphocyte-rich lesions. CONCLUSIONS: Our results indicate that monocytes from patients with angina are preactivated and express TF but CRP is unlikely to be a major priming factor in vivo. IFN and CRP further increase TF levels that may contribute to the hypercoagulable state in coronary disease.