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目的 研究血管性血友病因子 (vWF)Ala737→Glu突变对vWF功能的影响 ,分析vWFAla737→Glu突变致 2A型血管性血友病 (vWD)的分子病理机制。方法 用聚合酶链反应 (PCR)的方法对野生型的vWF全长表达质粒 (pSVvWF)进行体外定点诱变 ,在COS 7细胞中进行表达。分析其表达上清和细胞裂解液中的vWF∶Ag和上清的vWF的多聚物 ,观察患者血小板中的vWF多聚物及患者对DDAVP(糜凝 )的治疗反应。结果 vWFAla737→Glu的突变体上清vWF∶Ag是野生型的 76 .4% ,裂解液中vWF∶Ag是野生型的 98.8%。vWF突变体的多聚物形态和野生型相似 ,患者血小板中的vWF多聚物存在全部分子量的形式 ,糜凝治疗后vWF∶Ag明显升高。结论 vWFAla737→Glu的突变没有影响vWF的分泌和转运 ,vWFAla737→Glu突变致第Ⅱ类 2A型vWD。
Objective To investigate the effect of mutation of Ala737 → Glu of vWF on the vWF function and to analyze the molecular pathological mechanism of vWF induced by vWFAla737 → Glu mutation. Methods The wild-type vWF full-length expression plasmid (pSVvWF) was mutated in vitro by polymerase chain reaction (PCR) and expressed in COS-7 cells. Analyzes of their expression supernatants and polymers of vWF: Ag in supernatants and vWF in supernatants were performed to observe the therapeutic response of vWF polymers in platelets to DDAVP in patients. Results The mutant of vWFAla737 → Glu had a vWF: Ag of 76.4% of the wild type and the vWF: Ag in the lysate was 98.8% of the wild type. The vWF mutant has a multimer morphology similar to the wild-type, with vWF aggregates of all molecular weights present in platelets, with vWF: Ag significantly increased after migel treatment. Conclusion The mutation of vWFAla737 → Glu did not affect the secretion and transport of vWF, and the mutation of vWFAla737 → Glu caused the type Ⅱ 2A vWD.