特发性血小板减少性紫癜患者血小板相关抗体的表达及意义

目的探讨特发性血小板减少性紫癜（ITP）患者患病时PAIg的表达评价其在ITP中的临床意义。方法选取40例ITP患儿作为研究对象,同期门诊体检正常儿童20例为对照组,应用酶联免疫吸附法（ELISA法）测定血小板相关抗体（PAIgA、PAIgG）、采取单克隆抗体特异性俘获血小板抗原（MAIPA法）测定血小板特异性抗体抗血小板膜糖蛋白抗体（GPIIb/IIIa、GPIb/IX）,全自动血液分析仪测定血小板计数。结果 PAIgA、PAIgG、GPIIb/IIIa、GPIb/IX、血小板计数对照组分别为（4.22±0.78）ng/107PA、（23.17±3.46）ng/107PA、（0.33±0.01）A值、（0.31±0.01）A值、（137.82±42.37）×109/L,ITP组分别为（45.58±5.32）ng/107PA、（243.34±12.21）ng/107PA、（0.42±0.06）A值、（0.41±0.05）A值、（35.50±21.35）×109/L,两组以上各项指标之间比较均差异有统计学意义（P〈0.05）;ITP组血小板计数与特异性抗体GPIIb/IIIa、GPIb/IX之间具有负相关,相关系数分别为-0.24（P〈0.05）、-0.31（P〈0.05）。结论在ITP患儿中血小板相关抗体及特异性抗体均有明显升高,呈现阳性表达,ELISA法、MAIPA法联合检测有助于鉴别免疫性与非免疫性血小板减少型紫癜,能提高早期诊断率,并且有助于指导临床治疗。     

西洛他唑对糖尿病大鼠黏附分子的影响

目的　探讨西洛他唑对糖尿病 (DM)大鼠黏附分子CD54、CD10 6、CD62p的表达和坐骨神经传导速度及对肾脏以及主动脉病变的影响。方法　实验设正常组 8只大鼠 ,糖尿病组 8只大鼠 ,胰岛素组 7只大鼠和西洛他唑组 7只大鼠。测定各组坐骨神经传导速度 ,单个核细胞表面CD54、血小板表面CD62p以及肾脏、主动脉CD54或CD10 6的表达水平。结果　西洛他唑明显加快坐骨神经传导速度 (DM组 2 0 3m·s-1± 2 2m·s-1,西洛他唑组 2 8 9m·s-1± 7 9m·s-1,P <0 0 5 )。降低单个核细胞表面CD54的表达 (DM =6 5 1%± 14 9% ,西洛他唑组 2 5 4 %± 8 6 % ,P <0 0 5 ) ,减少肾脏和主动脉CD54和CD10 6表达 ,改善肾脏和主动脉病理变化。而降低血小板表面CD62p的作用不明显 (DM组 4 0 4 %±8 7% ,西洛他唑组 2 8 5 %± 3 7% ,P >0 0 5 )。结论　西洛他唑能降低CD54、CD10 6的表达 ,同时减缓糖尿病大鼠肾和主动脉及坐骨神经病变的发生发展     

免疫性血小板减少症患者外周血中microRNA-181a、microRNA-146a、microRNA-326及microRNA-142-3p的表达

目的 通过检测4种免疫相关的microRNAs （miR-181a、miR-146a、miR-326和miR-142-3p）在免疫性血小板减少症（ITP）患者外周血单个核细胞（PBMCs）中的表达,探讨miRNA在ITP中的作用及意义。方法 采用实时定量PCR（RT-PCR）法,检测34例ITP患者（ITP组）及31例健康人（正常对照组）外周血PBMCs中4种miRNAs的表达水平;改良MAIPA法检测ITP组中22例患者抗血小板自身抗体GPIIb/IIIa、GPIb/IX,分析miRNA与抗血小板自身抗体的关系,以及与血小板计数等临床指标的相关性。结果 ITP组PBMCs中miR-146a、miR-326和miR-142-3p表达水平与正常对照组相比明显降低（P<0.05）,miR-146a与miR-326 (r=0.437, P=0.011）,miR-146a与miR-181a (r=0.652, P<0.001),miR-326与miR-181a(r=0.745, P<0.001）的表达水平均呈显著正相关,且miR-146a与ITP患者血小板计数呈正相关（r=0.468,P=0.016）。ITP组不同性别间4种miRNAs表达水平差异无统计学意义（P>0.05）。ITP组中26例患者接受糖皮质激素治疗后,有效组与无效组4种miRNAs表达水平差异无统计学意义（P>0.05）。GPIb/IX、GPIIb/IIIa单阳性组、双阳性组和双阴性组间miRNAs的表达水平均无显著性差异（P>0.05）。结论 miR-146a、miR-326及miR-142-3p表达异常在ITP的发生和发展中可能发挥了一定的作用。     

vWF及GPIIb/IIIa受体在经皮冠状动脉腔内成形术后再狭窄中的作用

血管性血友病因子(von Willebrand factor, vWF)是由内皮细胞合成分泌的一种多聚糖蛋白.血小板膜糖蛋白(glucoprotein IIb/IIIa, GPIIb/IIIa)受体是血小板表面的一种糖蛋白.二者在介导血小板粘附、聚集及血栓形成等过程中起着重要作用.近年来对vWF和GPIIb/IIIa受体的结构功能进行了深入研究,表明vWF和GPIIb/IIIa受体的升高在经皮冠状动脉腔内成形术(percutaneous transluminal coronary angioplasty, PTCA)后再狭窄的发生方面具有重要的作用.现收集了最近几年来的有关资料,对vWF及GPIIb/IIIa受体在PTCA术后再狭窄中的作用综述如下.     

蛋白激酶C抑制剂对精氨酸升压素调控心脏成纤维细胞增殖及p27蛋白表达的影响

目的　观察蛋白激酶C(PKC)抑制剂白屈菜红 (chelerythrine)对精氨酸升压素 (AVP)介导的心脏成纤维细胞 (CF)增殖及p2 7蛋白表达的影响 ,探讨AVP促CF增殖的细胞内信号传导机制。方法　以培养的新生SD大鼠CF为实验模型 ,实验分 3组基础状态组 ;10 -7mol/LAVP组 ;10 -7mol/LAVP +10 -6mol/L白屈菜红组。每组 4只SD大鼠。采用胰酶消化、差速贴壁法培养CF ,四氮唑盐(MTT)比色法检测细胞增殖 ,碘化丙啶标记细胞DNA、p2 7蛋白的单抗和标记了FITC的二抗标记细胞内的p2 7蛋白 ,采用流式细胞分析仪 (FCM)技术测定细胞周期及p2 7蛋白表达的阳性率。结果　 ( 1)10 -7mol/LAVP和 10 -6mol/L白屈菜红共同作用 48h ,MTT比色法测定的CF的A值 ( 0 32± 0 0 1)明显低于 10 -7mol/LAVP组 ( 0 39± 0 0 1,P <0 0 1) ;( 2 )AVP与白屈菜红共同作用组CF的S期细胞百分率 ( 4 4%± 1 7% )和细胞增殖指数 ( 2 0 9%± 1 2 % )分别明显低于AVP组 ( 15 5 %± 1 4%和31 4%± 1 5 % ) ,而G0 /G1期细胞百分率 ( 79 1%± 1 2 % )明显高于AVP组 ( 6 8 6 %± 1 5 % ) ,两组间比较差异有非常显著意义 (P <0 0 1) ;( 3)AVP与白屈菜红共同作用组CF的p2 7蛋白表达阳性率( 91 7%± 2 2 % )明显高于AVP单独作用组 ( 6 3 3%± 1 9% ) ,二     

系统性红斑狼疮血小板减少患者巨核祖细胞分化障碍

目的　明确系统性红斑狼疮 (SLE)血小板减少患者骨髓巨核祖细胞增殖分化情况 ,以及患者血清对巨核祖细胞增殖分化的影响。方法　分离 7例SLE血小板减少患者 (SLE血小板减少组 )、11例胸外科开胸手术切除肋骨患者 (正常对照组 )和 4例SLE血小板正常但病情活动患者 (SLE血小板正常组 )的骨髓单个核细胞 ,体外巨核祖细胞 (CFU MK)无血清半固体培养 ;并分别加入SLE血小板减少和SLE血小板正常患者的血清 ,观察血清对CFU MK集落形成的影响。结果　SLE血小板减少组CFU MK集落数为 (2 7.33± 9.30 )个 /片 ,明显低于正常对照组的 (6 1.2 2± 2 9.71)个 /片和SLE血小板正常组的 (6 0 .0 0± 2 9.71)个 /片 (P值均 <0 .0 5 ) ;加入SLE血小板减少患者血清后 ,SLE血小板减少组 (n =7)和正常对照组 (n =7)CFU MK集落数分别减少为(14 .2 9± 6 .73)和 (2 9.4 4± 2 3.35 )个 /片 (P <0 .0 5 )。加入SLE血小板正常患者血清后 ,正常对照组 (n =7)CFU MK增加到 (115 .6 0± 72 .99)个 /片 ,与未加血清者的差异无显著性 (P <0 .0 5 ) ;而SLE血小板减少组 (n =7)无显著增加 (P >0 .0 5 )。结论　SLE血小板减少患者骨髓巨核祖细胞分化障碍 ,患者血清对骨髓巨核祖细胞的分化具有抑制作用     

β淀粉样蛋白1-40对大鼠皮层神经元凋亡的诱导作用

目的　观察 Aβ1 - 40 对大鼠皮层神经元的毒性作用。　方法　原代培养大鼠皮层神经元 ,分别采用AO- EB染色、TU NEL染色和 DNA凝胶电泳 ,观察不同终浓度的 Aβ1 - 40 对培养的神经元凋亡的诱导作用。　结果　在荧光显微镜下可见 ,经 AO- EB染色后凋亡的神经元胞核染色质着绿色荧光呈固缩状、圆珠状或新月状 ,Aβ1 - 40 三个浓度 (2 0 ,4 0和 80μg/ml)作用不同时间的凋亡率依次为 :2 4 h 2 0 .17%± 0 .76 % ,2 5 .17%± 3.82 % ,2 8.33±2 .84 % ;4 8h 2 2 .33%± 1.4 4 % ,38.83%± 1.2 6 % ,36 .6 7%± 1.0 4 % ;72 h 17.5 0 %± 1.80 % ,2 6 .5 0 %± 1.32 % ,30 .6 7%± 1.2 6 %。 TUNEL染色结果显示胞核内散在分布断裂的 DNA片段 ;琼脂糖凝胶电泳呈现典型的“梯状”带等细胞凋亡的特征性改变。　结论　 Aβ1 - 40 可诱导大鼠皮层神经元凋亡 ,以 4 0μg/ml作用 4 8h的诱导效果最为明显     

心力衰竭患者血小板功能状态研究

目的　探讨慢性充血性心力衰竭患者血小板功能状态。　方法　应用放射免疫法测定 4 3例充血性心力衰竭患者和 4 3例非心力衰竭对照组患者的血小板膜表面α-颗粒膜蛋白 (GMP14 0 )及纤溶酶原激活抑制物 (PAI-1 ) ,超声心动图检测左室舒张末期内径 (LVEDd)及左室射血分数 (LVEF)。　结果　心衰组GMP14 0 ( 3 3 .9± 2 .1 μg/L)、PAI-1 ( 0 .80± 0 .1 9AU/ml)明显高于对照组 ( 1 8.1± 4 9μg/L、0 .71±0 .9AU/ml) ,P值分别 <0 .0 0 1、<0 .0 5;IVEF( 51 .2 %± 1 0 .5% )明显低于对照组 ( 71 .3 %± 8.2 % ) ,P <0 .0 1。心衰组不同心功能患者的血小板功能状态不同 ,心功能越差 ,LVEDd越大 ,LVEF越小。　结论　慢性充血性心力衰竭时血小板高度活化 ,血液呈高凝状态。血小板活化程度可能与心衰的病程及愈后有关。     

血小板GPⅡb/Ⅲa拮抗剂RGDS和钙通道拮抗剂Nifedipine对GPIIb/IIIa活化的影响

目的：探讨GPⅡb/Ⅲa 拮抗剂RGDS和钙通道拮抗剂Nifedipine对ADP诱导的血小板GPⅡb/Ⅲa活化的影响。方法GP II b／11I a特异性抗体PAC-1标记活化GP II b／11I a,流式细胞仪检测静息及 ADP（5μmol/L）诱导的血小板PAC-1表达率。结果静息状态下的血小板PAC-1表达率为（0.80±0.85）％;在ADP（5μmol/L）激动下,PAC-1表达率为（77.27±9.47）%;RGDS以浓度依赖性的方式抑制PAC-1表达率,IC50值为206μmol/L;Nifedipine能以浓度依赖性的方式抑制PAC-1表达率,IC50值为178μmol/L;RGDS联合Nifedipine对PAC-1表达的联合抑制率为（60.15±16.35）％,二者的交互效应差异有统计学意义（P〈0.05）。结论GPIIb/IIIa受体拮抗剂RGDS和Ca2＋拮抗剂Nifedipine均抑制ADP诱导的血小板GPIIb/IIIa活化,其抑制作用呈浓度依赖性,随拮抗剂浓度增加抑制作用增强;两者对血小板GPIIb/IIIa活化的抑制存在协同作用。     

冰冻机采血小板超微结构回收率以及CD62P、PAC—I的改变

目的研究以终浓度5%二甲亚砜(DMSO)为主的冷冻保存剂加入机采血小板冻存1周、4周后血小板超微结构,体外回收率(RR)以及CD62P和PAC-Ⅰ的改变.方法跟踪30例机采血小板产品,采用全自动血细胞分析仪,透射电镜(TEM)技术和流式细胞术(FCM)等分析并比较加入以DMSO为主的冷冻保存剂在-120 ℃冻存1周,4周后血小板的超微结构,RR值及CD62P和PAC-Ⅰ的改变.结果 30例冷冻保存1周及4周后的血小板仍然具有相对完整的超微结构.冻存1周及4周后血小板的RR分别为冻前的79.49%±6.14%和74.07%±5.39%.新鲜机采血小板的CD62P及PAC-Ⅰ的值分别为50.72%±14.31%,53.23%±20.26%;冻存一周及四周后CD62P和PAC-Ⅰ的值分别为30.96%±11.02%,22.96%±9.90%;32.07%±9.21%,19.55%±10.33%.结论与新鲜机采血小板相比,冰冻血小板仍然具有较高的回收率和相对完整的超微结构,但也显示了不同程度的激活.与较高的回收率相对应, 冷冻保存1周及4周后的机采血小板产品仍然具有较高的CD62P及PAC-Ⅰ活性,其CD62P及PAC-Ⅰ值无显著性差异(P>0.05).     

活化血小板葡萄糖摄取及血小板膜整合素对其的影响

目的：了解活化血小板葡萄糖摄取过程及血小板膜整合素对其影响，探讨整合素与血小板能量代谢的关系，旨在研究血小板能量代谢涉及的信号传导，并为目前临床应用血小板膜糖蛋白Ⅱb/Ⅲa(GPⅡb/Ⅲa)单抗抗血小板活化提供新的理论依据。方法：用液态闪烁计数方法检测在血小板膜糖蛋白Ⅱb/Ⅲa单抗10E5及血小板整合素αvβ3单抗609干预与否情况下凝血酶激活后血小板摄取氚标2－脱氧葡萄糖[^3H]2-DG)率。结果：在生理浓度范围内活化血小板[^3H]2-DG摄取率较静息时明显升高，单抗10E5可充分阻断活化血小板葡萄糖摄取，单抗609可部分阻断。结论：整合素家族单抗可抑制血小板激活后葡萄糖摄取，提示其可能参与血小板能量代谢信号传导过程，并可能以GPⅡb/Ⅲa为主。     

Platelet Functions in Cardiopulmonary Bypass Surgery

Background

Haemorrhage after Cardio Pulmonary Bypass (CPB) Surgery is a well recognised complication that leads to significant morbidity and mortality. The incidence varies between 5-25% depending upon the clinical situation. Several factors are implicated as causative but none have been precisely proved.

Methods

Our study was an attempt to evaluate the haemostatic defect with particular reference to platelet function abnormalities during cardio pulmonary bypass surgery, in order to reduce the morbidity and mortality associated with post CPB haemorrhage. Flow cytometric evaluation of different platelet glycoproteins like GPIb/IX, GPIIb/IIIa and GMP-140 was done.

Results

The marker expression showed deregulation during surgery which returned to base after bypass was terminated. In contrast, the cases with bleeding showed significant variation. P-Selectin (GMP 140) expression decreased progressively till 3^rd post-operative day showing lack of activation of platelets in cases of severe bleeding.

Conclusion

Longer duration of CPB initiates plasmin generation through heparin, which raises the PAI-1-tPA complex and thereby down regulating the functions of platelets. This suggests a link between duration of CPB, bleeding, platelet dysfunction and fibrinolysis. Hence serial estimations of the levels of GMP-140 and tPA can predict severe bleeding.Key Words: CardioPulmonary Bypass, Platelet dysfunction, flowcytometry, platelet glycoproteins, haemorrhage     

Studies of human platelet ultrastructure and quantitative analyses of membrane glycoprotein IIB/IIIA in shear stress-induced platelet quick release reaction.

The ultrastructural characteristics of platelets and the quantitative analysis of membrane glycoprotein (GP) IIb/IIIa quick-release reaction induced by high shear stress (HSS) were studied with transmission electron microscope and immunocolloidal gold staining. The results showed that platelets were activated and aggregated by HSS (50 dynes/cm2 or 100 dynes/cm2). Besides, HSS also caused a special platelet quick-release reaction which was called granule-membrane-fused lytic release (GFLR). GFLR differed from releases I and II which were induced by ADP and thrombin respectively. Granules were released quickly and lysis occurred when all granules had been discharged in GFLR. GFLR is a special form of platelet quick release induced by HSS and is called release III by the authors. In the process of platelet activation and lysis, the gold particles of GPIIb/IIIa were increasingly condensed. The possible reasons of increased surface expression of GPIIb/IIIa stimulated by HSS are also discussed.

     

Effect of Xiaoyu Zhixue tablet on expression of platelet membrane glycoproteins in patients with hemorrhagic thrombopathy

Objective: To observe the effect of Xiaoyu Zhixue tablet (XYZXT) on the expression of platelet membrane glycoproteins in patients with hemorrhagic thrombopathy, and to explore its possible mechanism. Methods; The total of 148 patients with hemorrhagic thrombopathy were randomly divided into two groups, the traditional Chinese medicicne (TCM) group (n = 98) treated with XYZXT and the Western medicine (WM) group (n = 50) treated with adrenosin, vitamins C, K and P, both for 6 months. The therapeutic effect and the recovery rate of platelet aggregation in the two groups were observed. And platelet membrane glycoprotein (GP) Ib/IX, GPII b/IIIa complexes, GPIb, GPIIb, GPIIIa and P-selectin were analyzed by flow cytometry in both groups before and after treatment and also in 34 normal healthy subjects.Results: The total effective rate of hemostasis was 89.8% in TCM group and 54.0% in the WM group (X ²=45.83,P<0.01), and the recovery rate of platelet aggregation was 72.4% and 4.0% respectively (X ² = 62.06,P<0.01). The fluorescence intensity of GP I b/IX, GP II b/III a complexes, GPIb, GP IIIa and P-selectin were lower in both groups before treatment than those in the healthy subjects. Expression of above-mentioned marks was elevated in TCM group after 6 months’ therapy, which was insignificantly different as compared with the healthy subjects (P>0.05) and higher than those in the WM group (P<0.05).Conclusion: One of the mechanisms in treating hemorrhagic thrombopathy with XYZXT is that it could regulate the expression of GP I b/IX, GPII b/IIIa complexes, GP I b, GPIIIa and P-selectin at the level of receptor protein. Supported by the National Natural Science Foundation of China (No. 30171194)     

不同抗凝剂对血小板聚集和血小板膜糖蛋白的影响

目的探讨枸橼酸钠、肝素、乙二胺四乙酸（EDTA）对流式细胞术测血小板膜糖蛋白PAC-1和CD62P与二磷酸腺苷（ADP）和花生四烯酸（AA）血小板诱导聚集的影响。方法在不同抗凝剂情况下,采用血浆比浊法和流式细胞术观测70例健康志愿者血小板AA和ADP诱导聚集率和两个糖蛋白活化百分率。结果枸橼酸钠抗凝标本AA诱导血小板聚集率与肝素抗凝相当,ADP诱导聚集比肝素抗凝低（P〈0．05）,EDTA抗凝标本血小板几乎不聚;CTAD管抗凝标本所测的两个糖蛋白活化百分率最低,枸橼酸钠抗凝高,肝素抗凝活化百分率最高;EDTA抗凝CD62P活化百分率高（仅次于肝素）,PAC-1值很低,即EDTA对PAC-1表达是抑制的。结论枸橼酸钠为血小板聚集最佳抗凝剂;CTAD抗凝能最大限度防止血小板体外活化,是流式细胞术测血小板膜糖蛋白的抗凝最佳方案。     

冠状动脉性心脏病患者血小板表面膜糖蛋白的表达

目的研究冠状动脉性心脏病（coronary heart disease,CHD）患者血小板表面纤维蛋白原结合位点（PAC-1）、膜糖蛋白P-选择素（CD62P）、GPIIb11Ia（CD41a）和GPⅠb／Ⅸ（CD42b）的变化及临床意义。方法用流式细胞术（FCM）观察53例CHD患者及30名正常人PAC-1、CD62P、CD41a和CD42b表达。结果CHD患者PAC-1、CD62P、CD41a异常增高（P〈0．01）,CD42b降低（P〈0．05）。结论CHD患者伴有血小板活化,存在高凝状态病理基础,容易导致血栓的形成。     

融合蛋白TAP-SSL5对人血小板功能的影响

目的探讨抗炎抗凝双效融合蛋白TAP-SSL5对人血小板功能的影响。方法采用健康人富含血小板血浆以凝胶过滤法分离得到人血小板。流式细胞仪检测融合蛋白TAP-SSL5或小鼠抗人CD42b(glycoprotein Ibα,GPIbα)单克隆抗体(HIP1)与血小板的结合情况;以人血小板表面P-选择素的表达及PAC-1的结合反映血小板的激活程度;以全血电阻法定量分析TAP-SSL5对人血小板聚集功能的影响;为评价TAP-SSL5的出血风险,进一步观察了TAP-SSL5对小鼠尾部出血时间的影响。结果融合蛋白TAP-SSL5可与血小板结合,并竞争性抑制HIP1与血小板的结合,提示TAP-SSL5可与血小板表面的GPIbα结合,进而抑制GPIbα与vWF的相互作用。但高浓度的TAP-SSL5(终浓度30 mg/L)也能激活血小板,使人血小板表面P-选择素的表达增加(表达阳性率为90.4%)和PAC-1的结合量上升(阳性率为66.3%);终浓度为10、30 mg/L的TAP-SSL5可引起血小板的显著聚集。给小鼠单次尾静脉注射10 mg/kg的TAP-SSL5,可显著延长尾部出血时间,由对照组的(647.1±33.7)s延长至(753.6±127.3)s(P<0.01);而常规剂量组(3 mg/kg TAP-SSL5)尾部出血时间为(612.8±79.1)s,与对照组相比无显著差异(P>0.05)。结论融合蛋白TAP-SSL5保留了SSL5与血小板GPIbα结合的功能,有助于进一步提高TAP-SSL5的抗血栓作用,但同时也导致融合蛋白TAP-SSL5在高浓度情况下可延长出血时间和激活血小板。