人类血小板分子免疫学研究进展

自身免疫性血小板减少性紫癜(AITP)中血小板自身抗体通过其Fab段特异性结合血小板膜GP，CD5^ 和CD5^-B细胞共同参与AITP发病过程，慢性AITP患者自身抗原决定簇的数量有限，部分患者GP特异性自身抗体源于寡克隆B淋巴细胞增生。在AITP中通过阻断共刺激信号能够诱导血小板抗原特异性T细胞免疫耐受，其耐受机制为T细胞免疫无能。霉酚酸酯治疗激素耐药AITP的有效性与其对患者细胞和体液免疫功能的有效调节有关。血浆TPO水平测定有助于鉴别血小板破坏增多、骨髓增生低下或严重受抑所致的血小板生成减少，且对难治性贫血与慢性再生障碍性贫血的鉴别诊断具有重要意义。TPO检测可作为原发性与继发性血小板增多的实验室鉴别诊断指标。奎宁和肝素能够诱导药物相关自身抗体，引起血小板减少。PCR—SSP方法可用于新生儿HPA基因的产前检测及大规模研究不同人群HAP的基因分型。人类血小板糖蛋白上存在红细胞A、B、H血型抗原，A2亚型血小板可以作为O型血小板输注而不致引起抗血型A免疫反应。生物素可干扰标记血小板的功能。     

血小板特异性糖蛋白抗体和HLA抗体在特发性血小板减少性紫癜的表达

本研究旨在探讨特发性血小板减少性紫癜（ITP）中血小板特异性糖蛋白抗体和HLA抗体的表达。选择45例ITP患者,运用ELISA方法检测患者的血浆和血小板洗脱物血小板特异性糖蛋白抗体和HLA抗体。使用HPA分型试剂盒检测7个抗原系统HPA-1、2、3、4、5、6、15。结果表明,有45例患者检测到血清或者血小板洗脱物中存在血小板特异性糖蛋白抗体,其中以抗GPⅡb／Ⅲa／HIa和抗GpⅠb／Ⅸ最为常见;有11例患者同时表达HLA抗体和血小板特异性糖蛋白抗体。对病例37和40进行家系调查,发现这些病例所产生的血小板特异性糖蛋白抗体和HLA抗体与父母血小板抗原及HLA抗原不相合密切相关。结论：应用ELISA检测ITP病例的血浆和血小板洗脱物,并与病人的临床表现相结合,对提高ITP的诊断具有重要价值。     

特发性血小板减少性紫癜患者血小板对自身淋巴细胞的异常激活

目的　探讨特发性血小板减少性紫癜(ITP)患者血小板表面人类白细胞抗原 (HLA- DR)的表达和CD4+T淋巴细胞的活化状态。方法　采用流式细胞术检测 60例ITP患者和 60例正常人血小板表面HLA DR的表达水平和自身血小板活化的淋巴细胞产生白细胞介素 2(IL -2)水平。结果　ITP患者血小板表面HLA- DR的表达量为 0 .808%±0 .218%,正常对照组为 0. 025%±0. 019%,两者相比差异具有统计学意义(P<0. 01)。ITP患者CD4+T淋巴细胞经自身血小板激活后,IL 2的表达量为 5 .1% ±1 23%,较正常对照组(0 .0% )有明显差异(P<0 .01)。结论　ITP患者在自身血小板HLA DR抗原的作用下,出现了自身反应性T淋巴细胞的异常活化,T淋巴细胞处于高活性状态。     

特发性血小板减少性紫癜的细胞免疫机制

特发性血小板减少性紫癜 (idiopathicthrombocytopenicpur pura,ITP)是一种自身免疫性疾病 ,其特征是未成熟的血小板在单核巨噬系统被吞噬细胞破坏引起血小板减少症。越来越多的资料显示 ,除体液免疫机制外 ,在ITP尤其是慢性ITP(CITP)的发病中 ,细胞免疫起了非常重要的作用。ITP患者在T细胞亚群及功能、HLA系统、细胞因子、NK细胞和细胞凋亡等很多方面都可发现异常。1　T细胞亚群与功能异常　现已清楚B细胞产生自身抗体需要自身抗原特异性T细胞的参与。 1979年McMillan[1 ] 首先报道ITP时抗血小板自身抗体的产生需要T细胞及其…     

特发性血小板减少性紫癜患儿血小板数与T细胞凋亡率的相关性分析

特发性血小板减少性紫癜 (idiopathicthrombocytopaenicpurpura,ITP)是一种自身免疫性疾病 ,发病机制包括T、B细胞的异常活化和T细胞依赖性自身抗体的产生。以往的研究表明 ,ITP患者血小板的损伤主要与患者体内抗血小板相关抗体对血小板的损伤作用有关[1] 。近年来人们发现 ,ITP患者外周血激活的淋巴细胞增多、细胞凋亡不足与疾病的发生有关[2 ,3 ] 。我们采用流式细胞仪检测ITP患儿外周血T细胞凋亡率 ,全自动血细胞计数仪检测其血小板数 ,分析两者间的相关性 ,探讨T细胞凋亡异常在ITP血小板损伤中的作用。对象和方法1　研究对象　 …     

血小板抗体及淋巴细胞亚群检测在特发性血小板减少性紫癜中的诊断意义

目的 比较抗血小板特异性抗体、PAIgG及淋巴细胞亚群在特发性血小板减少性紫癜（ITP）及非免疫性血小板减少症中的水平,以评价其在ITP中的诊断价值.方法 用改良单克隆抗体特异性俘获血小板抗原法（MAIPA）检测患者血浆中抗血小板膜糖蛋白（GPⅡb/Ⅲa、GPⅠb和P-选择素）的特异性抗体.利用流式细胞术（FCM）检测患者外周血中PAIgG及淋巴细胞亚群.结果 ITP组MAIPA的阳性率为63.3%,非免疫性血小板减少组为阴性;PAIgG分别为73.3%、45%.淋巴细胞亚群中,ITP组CD3、CD4、CD4/CD8显著高于正常对照组,CD8、CD19则显著低于正常对照组.结论 抗血小板特异性自身抗体抗体对提高ITP的诊断有一定的实用价值,淋巴细胞亚群的变化能较好地反映ITP 的病理机制.     

多种血小板特异性抗体联合检测在特发性血小板减少性紫癜诊断中的价值

特发性血小板减少性紫癜 (ITP)是一类由体液和细胞免疫异常导致血小板破坏增多的疾病[1,2 ] 。血小板自身抗体对ITP的诊断及探讨ITP的发病机制具有重要意义。我们采用单克隆抗体 (单抗 )特异性俘获血小板抗原 (MAIPA)技术并加以改进 ,同时检测了ITP患者 5种血小板糖蛋白(GP)特异性抗体 ,评价其对ITP的诊断价值。病例和方法1　病例　选择我院 2 0 0 2年 3月至 2 0 0 3年 3月ITP患者30例。其中男 12例 ,女 18例 ,年龄 6～ 6 2岁。所有待测者均未接受过血小板输注 ,采集标本时血小板数为 (5～ 6 0 )×10 9/L。采集患者外周血 5ml,5 …     

抗原呈递细胞与T及B淋巴细胞异常在特发性血小板减少性紫癜发病机制中的作用

特发性血小板减少性紫癜(ITP)是一种自身免疫性疾病,发病机制尚未完全明确.目前研究证实,ITP患者体内存在抗原呈递细胞(APC)与T及B淋巴细胞异常.笔者拟就APC与T及B淋巴细胞异常,以及3者间相互作用在ITP发病机制中的作用进行综述,为进一步了解ITP发病机制提供参考.     

T细胞异常与特发性血小板减少性紫癜

80年代以来，越来越多的研究证实特发性血小板减少性紫癜(ITP)患者体内存在着多种T淋巴细胞异常，T淋巴细胞表型及功能的异常可能在ITP发病中起重要作用。现就ITP患者T淋巴细胞异常及可能的致病机制作一综述。     

特发性血小板减少性紫癜患者血小板相关抗体和淋巴细胞亚群的临床观察

目的 检测急性特发性血小板减少性紫癜患者血小板相关抗体(PAIg)和淋巴细胞亚群,探讨其在特发性血小板减少性紫癜(ITP)免疫发病机制中的作用及临床意义.方法 分别采用酶联免疫吸附法(ELISA)和流式细胞术(FCM)检测20例ITP患者及20例健康人外周血中PAIg 和T淋巴细胞表型.结果 ITP患者组PAIg明显高于健康人组(P＜0.01);ITP患者组CD3 、CD4 、CD4 /CD8 显著低于健康人组(P＜0.01),CD8 显著高于健康人组(P＜0.01).结论 血小板相关抗体和T淋巴细胞亚群的变化能较好的反映ITP发病的病理机制.     

Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa.

The object of this study was to further localize autoantigenic structures on IIb-IIIa and, if possible, to precisely identify the epitopes recognized by human autoantibodies. In this paper, we identify a 50-kD chymotryptic fragment of IIIa that is recognized by a high percentage of human autoantibodies, typified by the prototype IgG autoantibody RA, which binds to IIIa on intact platelets as well as in an immunoblot assay under nonreduced conditions. Using an immunoblot assay, a carboxy-terminal region of this fragment (33 kD) that contains the cysteine-rich domains of IIIa was found to carry the epitope(s) recognized by the prototype autoantibody RA. The amino-terminal amino acid sequence of the reduced 33-kD fragment, the smallest fragment that retains the RA epitope, is XPSQQDEXSP, and that of the reduced 50-kD fragment is IVQVTFD. This indicates that the 33-kD fragment consists of approximately 175 amino acids beginning at residue 479 and extending at least through residues 636-654, while the 50-kD fragment spans the same region but begins at residue 427. It is apparent that the 33-kD fragment is generated from the 50-kD fragment by additional chymotryptic hydrolysis but remains associated because of the multiple disulfide bonds that are characteristic of this cysteine-rich domain. Sera from 48% of patients with chronic ITP and 2 of 8 patients with acute ITP contain antibodies that bind to the 50-kD fragment in an ELISA. Antibodies of the same specificity are also found in one-third of patients with either secondary immune thrombocytopenia or apparent non-immune thrombocytopenia. We conclude that the 50-kD cysteine-rich region of IIIa is a frequent target of autoantibodies in ITP, but that such antibodies may also be present in cases of thrombocytopenia that cannot be linked to an apparent autoimmune process.     

Idiopathic thrombocytopenic purpura

Idiopathic(autoimmune) thrombocytopenic purpura(ITP) is characterized by thrombocytopenia and normal to increased numbers of megakaryocytes. ITP is a disease caused by circulating autoantibodies that react with the platelet membrane. It is thought that platelet-associated IgG(PAIgG) plays an important role in the mechanism of ITP, since increases in PAIgG are closely related to decreases in platelet count in patients with this disease. However, it is possible that PAIgG includes immune complex and platelet antibodies to human platelet alloantigens(HPA) other than autoantibodies. There have been several recent reports on autoantibodies to glycoprotein IIb/IIIa and Ib/IX. Although the etiology of ITP remains unclear, both genetic and environmental factors appear to be involved in it. With respect to the genetic aspects of ITP, the human leukocyte antigen (HLA) haplotype of patients is considered a potentially important factor in etiology. Various methods have been used for the treatment of chronic ITP. However, splenectomy and corticosteroids are still the mainstays of therapy. Because of the heterogeneity of the disease, however, approximately 20% of cases are refractory to these treatments. New therapy of chronic or refractory ITP with thrombopoietin, antiCD40 ligand antibody, and Helicobactor pylori eradication have recently been reported.     

血小板糖蛋白特异性抗体在骨髓增生异常综合征患者中的表达及其意义

本研究探讨血小板糖蛋白特异性抗体在骨髓增生异常综合征（MDS）患者发病中的作用。采用改良的MAIPA法检测血浆血小板糖蛋白特异性抗体（抗GPⅡb／Ⅲa抗体和抗GPIb／Ⅸ抗体）。若患者的OD值大于正常OD值的均数＋3倍标准差则为阳性。结果表明：MDS组总阳性率为16．67％（5／30），ITP组总阳性率为46．67％（14／30），两者之间差异有显著性（P〈0．05）。结论：部分MDS患者的糖蛋白特异性抗体为阳性，这表明部分MDS患者的血小板减少与免疫因素有关；血小板糖蛋白特异性抗体所致的血小板破坏在MDS患者的血小板减少中可能具有一定作用，这为MDS患者的免疫抑制剂治疗提供了新的依据。     

Platelet autoantibodies in immune thrombocytopenic purpura.

In summary, the search for a useful clinical laboratory diagnostic assay for the antiplatelet antibodies has been long and difficult. Measurement of platelet associated IgG (PAIgG) has been disappointing as a way to detect autoantibodies. This is primarily due to the fact that platelets normally contain IgG in their alpha granules in an amount that varies with plasma IgG levels and age of the platelets. Furthermore, the amounts of platelet associated IgG is affected by the presence of circulating immune complexes, platelet activation, and drug dependent antibodies. The newer, platelet antigen capture techniques are promising, but further testing will be needed to confirm their value to the clinician. Methods that allow incubation of patient serum or plasma with intact platelets (MAIPA and immunobead) have greater sensitivity than techniques in which the patient antibody is tested against previously isolated platelet glycoproteins. These assays are currently available in a only a limited number of platelet immunology laboratories. Platelet autoantibodies are directed against a number of glycoprotein antigens on the platelet surface. Most studies have shown that anti GPIIb/IIIa antibodies are the most common, although antibodies against GPIb/IX and other targets are frequently detected. Many patients have multiple antiplatelet antibodies circulating simultaneously. The clinical significance of antibodies with different specificity is under investigation. The precise epitopes on GPIIIa that bind antiplatelet autoantibodies have been studied to a limited extent. Some investigators report that the vast majority of platelet antigens are conformation dependent, being destroyed by treatment with EDTA (separation of GPIIb and GPIIIa) or denaturation with detergents. Others report sequence specific peptide antigens. Further investigation promises to better define the targets for platelet autoantibodies; improved clinical management of patients with ITP is the long term goal of these studies.     

The detection methods for antiplatelet autoantibodies (phase II and phase III assay)

A variety of methods are utilized to detect antiplatelet autoantibodies nowadays, platelet-associated immunoglobulin G(PAIgG) is a phase II assay of limited value in the meaning of its sensitivity and specificity for the diagnosis of idiopathic thrombocytopenic purpura(ITP), although it has been ordered in many occasions. The newer antigen-specific assays(phase III), which can identify autoantibodies against platelet glycoprotein(GP)s, such as GP IIb/IIIa, with greater specificity but lower sensitivity are rarely performed in clinical situations in Japan. Development of novel systems to detect clinically more relevant markers, including specific antiplatelet autoantibodies, is necessary for the diagnosis of ITP.     

血小板特异性自身抗体检测在特发性血小板减少性紫癜中的意义

目的 检测特发性血小板减少性紫癜(ITP)患者体内的血小板特异性自身抗体与临床严重程度和临床疗效间的关系. 方法 应用改良的单克隆抗体特异性俘获血小板抗原(MAIPA)法检测血小板膜糖蛋白(GPⅡ bⅢa、GPIb)特异性自身抗体. 结果 ITP组(40例),10例为单一抗GPⅡ b Ⅲa抗体阳性.6例为单一抗GPIbα抗体阳性,20例为双抗体阳性,4例为双抗体阴性.非免疫性血小板减少组与正常对照组均为阴性.抗GPⅡbⅢa抗体(b=-0.071,P<0.01)、抗GPlbα抗体(b=-0.092,P<0.01)均与采血时血小板计数呈显著负相关.治疗后,双抗体阳性组8例治疗无效,单抗体阳性组(16例)1例治疗无效(χ2=6.09,P<0.05). 结论 血小板特异性自身抗体检测对鉴别特发性和非免疫性血小板减少症有一定价值.抗体种类与临床疗效有一定关系.     

Autoepitopes and alloepitopes of type IV collagen: role in the molecular pathogenesis of anti-GBM antibody glomerulonephritis

Anti-glomerular basement membrane (anti-GBM) antibodies elicited by autoimmune or alloimmune mechanisms are associated with aggressive forms of rapid progressive glomerulonephritis. Pathogenic anti-GBM autoantibodies and alloantibodies target the noncollagenous (NC1) domains of the alpha3alpha4alpha5(IV) collagen, a major GBM component. In autoimmune anti-GBM glomerulonephritis, a breakdown of immune self-tolerance leads to the activation of autoreactive B and T cells recognizing epitopes within the alpha3NC1 subunit. In the GBM, the conformational epitopes targeted by anti-GBM autoantibodies are structurally sequestered within the alpha3alpha4alpha5NC1 hexamer complex formed upon assembly of collagen IV chains into trimeric molecules and networks. Autoantibodies selectively bind to and dissociate a subset of alpha3alpha4alpha5NC1 hexamers composed of monomer subunits, whereas hexamers containing NC1 dimer subunits are resistant to dissociation by autoantibodies. The crypticity of alpha3NC1 autoepitopes suggests that self-tolerance to alpha3(IV) collagen is broken by structural alterations of the native alpha3alpha4alpha5NC1 hexamer that unmask normally sequestered epitopes, triggering an autoimmune reaction. Post-transplant anti-GBM nephritis in the renal allograft of transplanted Alport patients is mediated by an alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen, present in the allograft GBM but absent from Alport basement membranes. Alloantibodies from patients with autosomal-recessive Alport syndrome predominantly bind to the alpha3NC1 domain, whereas alloantibodies from X-linked Alport patients target preferentially, though not exclusively, epitopes within the alpha5NC1 subunit. The accessibility of the alloantigenic sites within the alpha3alpha4alpha5NC1 hexamers, contrasting with the crypticity of autoantigenic sites, suggest that different molecular forms of alpha3alpha4alpha5(IV) collagen initiate the immunopathogenic responses in the two forms of anti-GBM disease. Advances in elucidating the structure of the GBM antigen and the identification of the pathogenic B and T cell epitopes, along with new insights into the pathogenic mechanisms at cellular and molecular level will facilitate the development of targeted strategies for prevention, detection, and treatment of human anti-GBM antibody glomerulonephritis.     

HLA-DRB1等位基因与儿童特发性血小板减少性紫癜

目的：研究人类白细胞抗原（LA）DRB1等位基因与儿童特发性血小板减少性紫癜（ITP）的关系。方法：用PCR－SSO法对42例ITP患儿进行HLA－DRB1等位基因分型，同时用改良的血小板抗原单抗特异性固相化法（MAIPA）检测其中36例ITP患儿血清中的抗GPⅡb/Ⅲa和GPⅠb/Ⅸ自身抗体。结果：（1）与健康对照相比，ITP患儿HLA－DRB1＊17基因型显著升高（P＜0．05，RR＝2．76，EF＝0．1064），而HLA－DRB1＊1202基因型显著降低（P＜0．025，RR＝0．20，PF＝0．7616）。（2）慢性难治性ITP患儿与非难治性患儿相比，HLA－DRB1＊11基因型显著升高（P＜0．025），且具有DRB1＊11的患儿主要（5／6）为女性年长患儿；（3）抗GPⅡb/Ⅲa及抗GPⅠb/Ⅸ自身抗体的阳性率都与HLA－DRB1＊02（15／16）基因型显著相关（P分别为0．02和0．01），但难治性和非难治性ITP患儿间抗体阳性差异无显著性（P＞0．1）。结论：（1）DRB1＊17可能与儿童ITP的易感性有关，而DRB1＊1202则可能对儿童ITP的发病具有保护作用；（2）具有DRB1＊11基因型的患儿易发展为慢性难治性ITP；（3）血小板自身抗体与抗原表位的反应可能受DRB1＊02限制，但自身抗体阳性与否并不能预示ITP患儿的预后。     

Diagnosis of idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP) has an incidence of 5.8-6.6 per 100,000 adults and represents a frequent cause of impaired cellular haemostasis in clinical practice. Its diagnosis is still one of exclusion, because the diagnostic value of proposed biological markers of the disease has been disputed. The potential contribution of biomarkers both of the autoimmune reaction (leptin, free and cell-bound anti-platelet autoantibodies, specific B cells) and of thrombopoiesis (bone marrow histology, thrombopoietin, glycocalicin, reticulated platelets) to the diagnosis of ITP will be discussed. There is evidence that some of these biomarkers indeed could be useful in the diagnosis of ITP. To cope with the rapid progress in ITP therapy, prospective studies on well characterized cohorts are necessary to allow an efficient and definite diagnosis of this disease.     

特发性血小板减少性紫癜患者脾脏CD5+和CD5-B细胞共同参与血小板自身抗体的产生

目的：研究慢性特发性血小板减少性紫癜（ITP）患者脾脏CD5^ B细胞水平的变化及CD5^ 和CD5^-B细胞与血小板膜糖蛋白（GP）特异性自身抗体产生的关系，以识别致病B细胞亚群。方法：应用双色流式细胞仪检测8例慢性ITP患者脾脏CD5^ B细胞水平。选择4例血浆抗GPⅡb/Ⅲa和抗GP Ⅰb/Ⅸ抗体双阳性ITP切脾患者，应用Ficoll密度梯度离心及花环形成分离法分离脾脏B淋巴细胞，继而采用镝产珠分选法分选、纯化CD5^ B细胞和CD5^-B细胞，并分别进行体外培养，应用改良MAIPA法检测血浆和细胞培养上清液的血小板特异性抗体。结果：ITP患者脾脏CD5^ B细胞水平圈晨自身免疫性疾病患者略有增高，二者之间差异无统计学意义。CD5^ B细胞水平与患者血小板计数无相关性。4例血浆抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。另外1例CD5^ B细胞培养液抗GPⅡb/Ⅲa抗体阴性，抗GPⅠb/Ⅸ抗体阳性;CD5^-B细胞培养液抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。结论：脾脏CD5^ 和CD5^-B细胞 均可产生血小板GP特异性自身抗体，抗体产生种类和滴度无明显差异。提示二者共同参与了ITP的发病过程。