论文部分内容阅读
目的确定1例大疱性类天疱疮并获得性血友病 A 患者的临床和实验室诊断;通过体内外干预实验观察获得性血友病 A 患者体内的 FⅧ抑制物对正常人血浆及兔 FⅧ凝血活性(FⅧ:C)的影响;确定患者 FⅧ抑制物的 TgG 亚型和结构表位,以揭示该病发病的分子机制。方法用Ⅰ期法测定患者血浆 FⅧ:C;以 Bethesda 单位(BU)表示 FⅧ:C 抑制物滴度;用蛋白 A-琼脂糖柱纯化患者及正常人血浆 IgG;用活化部分凝血活酶时间(APTT)分析其对 FⅧ:C 的抑制作用;观察 FⅧ抑制物对兔血浆 FⅧ:C 活性的抑制作用;分别用抗 IgG_1、IgG_2、IgG_3、IgG_4抗体作 Western blot,结合灰度扫描确定患者血浆 IgG 亚型相对表达量;散射比浊法测定患者及正常人体内各 IgG 亚型浓度;用 FⅧ/FⅧ抑制物固相结合试验及 Western blot 鉴定患者 IgG 抗体(FⅧ抑制物)作用于 FⅧ的具体“表位”。结果①患者 APTT 明显延长,正常血浆不能纠正;FⅧ:C<1.5%,FⅧ抑制物滴度为147.8 BU;②纯化的患者 IgG 以剂量依赖方式抑制正常人混合血浆 FⅧ:C;动物实验显示患者血浆能以时间依赖方式延长兔血浆 APTT;③Western blot 结果显示 FⅧ抑制物成分主要为 IgG_4,其次为 IgG_1,FⅧ抑制物作用于FⅧ:C 的结构相对分子质量为44×10~3。散射比浊法测定结果显示患者体内 IgG_4、IgG_1含量明显高于正常人。结论研究结果证明大疱性类天疱疮并获得性血友病 A 患者体内 FⅧ抑制物对 FⅧ:C有抑制作用,FⅧ抑制物的 IgG 亚型主要为 IgG_4,其次为 IgG_1;患者 FⅧ抑制物作用于 FⅧ的表位为相对分子质量为44×10~3的肽段,揭示了该获得性血友病 A 发生、发展的分子机制。
Objective To determine the clinical and laboratory diagnosis of bullous pemphigoid and acquired hemophilia A. To observe the effect of FⅧ inhibitor in patients with acquired hemophilia A on normal human plasma and rabbit F Ⅷ coagulation activity (F Ⅷ: C); To determine the Tg F subtype and structure epitopes in patients with inhibitors to reveal the molecular pathogenesis of the disease. Methods Plasma F Ⅷ: C was measured by phase Ⅰ method. The titer of F Ⅷ: C inhibitor was measured by Bethesda unit (BU). The plasma IgG was purified by protein A-Sepharose column and the activity of activated partial thromboplastin time The inhibitory effect of FⅧ inhibitor on the activity of FⅧ: C in plasma was observed by APTT assay. Western blot was performed with anti-IgG1, IgG_2, IgG_3 and IgG_4 antibodies respectively. The plasma IgG FⅧ / FⅧ inhibitor solid phase binding assay and Western blot were used to determine the effect of IgG antibody (FⅧ inhibitor) on FⅧ specific “ gauge”. Results ① The patient’s APTT prolonged significantly, the normal plasma could not be corrected; the FⅧ: C <1.5%, the FⅧ inhibitor titer was 147.8 BU; ② The purified IgG inhibited the normal human mixed plasma FⅧ: C in a dose-dependent manner; Plasma prolongation of plasma APTT in a time-dependent manner; ③Western blot results showed that the components of FⅧ inhibitor were mainly IgG_4, followed by IgG_1, and the FⅧ: C FⅧ: C structure relative molecular mass was 44 × 10 ~ 3. The results of nephelometry showed that the content of IgG_4 and IgG_1 in patients was significantly higher than that in normal people. Conclusions The results of the study demonstrate that FⅧ inhibitor inhibits F Ⅷ: C in patients with bullous pemphigoid and acquired hemophilia A. The IgG subtype of FⅧ inhibitor is mainly IgG_4 followed by IgG_1. Patients with FⅧ inhibitor The epitope that acts on F Ⅷ is a peptide with a relative molecular mass of 44 × 10 ~ 3, revealing the molecular mechanism of the development and progression of acquired hemophilia A.