摘要
抗磷脂综合征(antiphospholip id syndrome,APS)是一种非器官特异性自身免疫性疾病,其特征为血清抗磷脂抗体(antiphosphoslipid antibody,APA)阳性,临床上有静脉或动脉血栓形成、反复妊娠丢失、血小板减少等临床症状。APS可引起多种不良妊娠,如习惯性流产、先兆子痫、胎盘功能不全、胎儿生长受限(fetalgrowth restriction,FGR)、早产,因此成为近年来产科研究的热点。APA导致不良妊娠结局的原因是多方面的,其中血栓形成是一个主要因素。而血管内皮细胞受损伤或激活被认为是血栓形成的首要因素。内皮细胞的活化可表现在许多方面,其中粘附分子表达增加是一个重要的方面。国外许多研究发现抗磷脂抗体可以激活内皮细胞,使粘附分子表达升高。但国内未见报道。
由APA引发的内皮细胞活化具有特异的信号转导途径,并非通过简单的磷脂结合途径。β_2GP I是APS重要的自身抗原,大多数的APA都是识别β_2GP I,故认为内皮细胞膜表面存在着β_2GP1的受体。近年来国外研究证实,β_2GP I可以通过内皮细胞膜表面的Annexin A2与磷脂发生结合,认为内皮细胞膜上的Annxin A2作为β_2GP I的受体,发挥促进内皮细胞活化的作用。
本文分为三个部分:第一部分为体外培养脐静脉内皮细胞(HUVEC),采用实时荧光定量RT-PCR和流式细胞技术检测APS患者血清诱导HUVEC细胞间粘附分子-1(ICAM-1)和E选择素(E-selectin)表达的变化,探讨APS患者是否存在内皮细胞活化。第二部分探讨Annexin A2在APS血管内皮细胞活化中的作用,发现抗Annexin A2抗体可通过非Fc受体途径活化内皮细胞,作为活化受体的Annexin A2可能需交联或聚簇,才能引起内皮细胞活化,抗Annexin A2抗体Fab片断及内皮细胞Annexin A2 siRNA干扰均可阻碍APS血管内皮活化。第三部分通过研究氟伐他汀对APS患者血清诱导HUVEC ICAM-1表达的影响,探讨氟伐他汀是否可因为影响APS内皮活化而成为治疗APS的一种新的选择药物。
目的:观察抗磷脂综合征患者血清对血管内皮细胞表达ICAM-1和E-selectin的影响。
方法:用采集的10例APS患者和10例健康对照者血清分别处理体外培养的脐静脉内皮细胞,用荧光定量PCR检测脐静脉内皮细胞上细胞间粘附分子-1(ICAM-1)和E-选择素(E-selectin)mRNA表达,用流式细胞仪检测二者蛋白的表达。
结果:APS血清处理后脐静脉内皮细胞上粘附分子ICAM-1和E-selectinmRNA和蛋白均比对照组明显升高,差异有显著性(P<0.05)。
结论:抗磷脂综合征患者血清可活化血管内皮细胞,使粘附分子表达增加。
目的:探讨Annexin A2在抗磷脂综合征血管内皮活化中的作用。
方法:用抗Annexin A2抗体及其抗体片断分别处理体外培养的脐静脉内皮细胞,用荧光定量PCR和流式细胞仪分别检测脐静脉内皮细胞上细胞间粘附分子-1(ICAM-1)mRNA和蛋白表达。再用抗Annexin A2抗体Fab片断或AnnexinA2 siRNA对APS血清组脐静脉内皮细胞进行干预,用荧光定量PCR和流式细胞仪分别检测脐静脉内皮细胞上ICAM-1 mRNA和蛋白表达变化。
结果:抗Annexin A2抗体和抗Annexin A2抗体F(ab')_2片断均能引起脐静脉内皮细胞上粘附分子ICAM-1 mRNA和蛋白表达增高,而抗Annexin A2抗体Fab不能。经Fab预处理的脐静脉内皮细胞,再经抗Annexin A2抗体或APS血清作用时,ICAM-1 mRNA和蛋白表达较处理前减少。转染Annexin A2 siRNA的脐静脉内皮细胞,APS血清作用时,ICAM-1 mRNA和蛋白表达也较转染前减少。
结论:抗Annexin A2抗体通过非Fc途径活化内皮细胞。作为活化受体的Annexin A2可能需交联或聚簇,才能引起内皮细胞活化,抗Annexin A2 Fab可阻碍APS内皮细胞活化.对内皮细胞进行Annexin A2 siRNA干扰也可阻碍APS内皮细胞活化。
目的:研究氟伐他汀对抗磷脂综合征血管内皮活化的影响。
方法:用APS患者血清、健康对照血清及培基分别处理人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC),其中APS血清处理组预先加入不同浓度的氟伐他汀(0、2.5、5和10μmol/L)进行干预,用实时荧光定量PCR检测脐静脉内皮细胞上细胞间粘附分子-1(intercellular adhesionmolecule,ICAM-1)mRNA表达,用流式细胞仪检测其蛋白的表达。
结果:与对照组相比,APS患者血清可诱导ICAM-1 mRNA和蛋白的表达增加,而氟伐他汀干预对ICAM-1mRNA和蛋白表达有不同程度抑制,且呈浓度依赖性(P<0.05)。
结论:氟伐他汀可减少脐静脉内皮细胞粘附分子表达,影响抗磷脂综合征患者血管内皮活化。提示他汀类药物可为APS的治疗提供新的选择。
Antiphospholipid syndrome(APS) is a non-organ specific autoimmune disease manifested by recurrent fetal loss,arterial occusions,repeated venous thrombosis and thrombocytopenia.Obstetrical features presently include recurrent recurent spontaneous abortion,preeclampsia,intrauterine growth restriction,placental disfuction, premature labour,and much attention was payed to APS in obstetric field.Severe pregnancy complications may be attribute to thrombosis. Endothelial cell activation is the principal reason of thrombosis,and can be measured by expression of endothelial cell adhesion molecules. Several overseas studies have demonstrated that aPL antibodies activate endothelial cells(ECs) in vitro,as demonstrated by enhanced expression of adhesion molecules,but domestic studies is fewer.
Binding of APA to these cells has been assumed to result from its interaction with membrane phospholipids.β_2-GP1has been found to be an important autoantigen in the antiphospholipid antibody syndrome.It is now generally accepted that most antiphospholipid antibodies recognizeβ2GP I bound to the cardiolipin-coated microplates used in clinical“anticardiolipin”assays。there is said to be a receptor ofβ_2-GP1 on endothelial cells surface.Recent overseas studies indicated e that annexin A2 mediates the binding ofβ_2-GP1 to endothelial cells,annexin A2 is the receptor ofβ_2-GP1on endothelial cells surface for Endothelial cell activation.
There are three part in the paper.The first part was about investigating the effects of umbilical vein endothelial cells activation by the serum of patients with antiphospholipid syndrome.HUVECs were cultured in vitro,and treated with APS serum,and adhesion molecule ICAM-1和E-selectin mRNA was measured by real-time fluorescent quantitative-polymerase chain reaction and its antigen by flow cytometry.the second part was about investigating the effects of Annexin A2 mediates endothelial cell activation in antiphospholipid syndrome. We found anti-annexin A2 antibodies cause endothelial cell Activation viia non-Fc receptor pathway.These observations suggest a novel pathway for endothelial activation in APS that is initiated by crosslinking or clustering of annexin A2 on the endothelial surface. anti-annexin A2 monomeric Fab can block endothelial activation, treat by annexin A2 RNA interference also can block endothelial activation.the third part was about investigating whether fluvastatin interfering with ICAM-1 expression of HUVECs treated by APS serum,so that blocked endothelial cell activation in APS.we may provide a rationale for using statins as a therapeutic tool in treatment of APS.
Objective:To investigate the effects on expression of ICAM-1 and E-selectin in vascular endothelial cells induced by by the serum of patients with antiphospholipid syndrome
Methods:The serum was collected from 10 patients with APS and 10 normal controls,Human umbilical vein endothelial cells(HUVEC) were treated with the two kind of serum respectively.Adhesion molecule ICAM-1和E-selectin mRNA was measured by real-time fluorescent quantitative-polymerase chain reaction and its antigen by flow cytometry.
Results:APS serum significantly increased the expressions of ICAM-land E-selectin on HUVEC-12 were from mRNA levels and protein level,compared with control serum(P＜0.05).
Conclusious:umbilical vein endothelial cells can be activated by APS serum,and increased expression of adhesion molecules.
Chapter 2
Annexin A2 mediates endothelial cell activation in antiphospholipid syndrome
Objective:To investigate the effects of Annexin A2 mediates endothelial cell activation in antiphospholipid syndrome.
Methods:Human umbilical vein endothelial cell(HUVEC) were treated with anti-annexin A2 antibodies and anti-annexin A2 fragments F(ab')2、F(ab),and adhesion molecule ICAM-1 mRNA of HUVEC was measured by real-time fluorescent quantitative-polymerase chain reaction and its protein by flow cytometry.Then HUVEC in APS serum group was pretreated with anti-annexin A2 F(ab) or Annexin A2 siRNA,and adhesion molecule ICAM-1mRNA of HUVEC was measured by real-time fluorescent quantitative-polymerase chain reaction and its protein by flow cytometry.
Results:Incubation of HUVEC with anti-annexin A2 antibodies and bivalent anti-annexin A2 F(ab_)2 fragments both led to a higher ICAM-1 expression in mRNA and protein,but incubation of HUVEC with monomeric Fab couldn't do it. HUVEC p retreated with anti-annexin A2 Fab significantly reduced the ICAM-1 response to anti-annexin A2 antibodies or APS serum.HUVEC pretreated with Annexin A2 RNA interference also significantly reduced the ICAM-1 response to APS serum.
Conclusious:Anti-annexin A2 antibodies cause endothelial cell activation via non-Fc receptor pathway.These observations suggest a novel pathway for endothelial activation in APS that is initiated by crosslinking or clustering of annexin A2 on the endothelial surface.Anti-annexin A2 monomeric Fab can block endothelial activation in APS,and annexin A2 RNA interference can also do it.
Objective:The present study was undertaken to investigate whether fluvastatin interferes with endothelial cell activation induced by the serum of patients with antiphospholipid syndrome.
Methods:human umbilical vein endothelial cells were treated with serum from APS patients or normal controls or with medium alone,cells were pretreated with fluvastatin(0、2.5、5 and 10μmol/L)before treated with APS serum.Adhesion molecule ICAM-1mRNA was measured by real-time fluorescent quantitative -polymerase chain reaction and its antigen by flow cytometry.
Results:Incubation of EC with APS serum led to a higher ICAM-1 expression compared with incubation with control serum,fluvastatin treatment of the cells significantly reduced the ICAM-1 response to APS serum,and this effect was dose dependent(P＜0.05).
Conclusions:fluvastatin exerts a protective effect towards APS serum at the EC level by decreasing leukocyte adhesion molecule expression on endothelial cells.our data provide a rationale for using statins as a therapeutic tool in treatment of APS.
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