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目的 研究在流体切应力作用下表达突变的细胞与固着的血管性血友病因子 (vWF)相互作用中GPⅠbα突变 (A15 6V)的意义。方法 在GPⅠbαcDNA中直接诱发的突变克隆到哺乳类表达载体pDX的EcoRⅠ位点 ,随后将突变的cDNA转染在CHOβⅨ细胞。人的vWF通过甘氨酸和氯化钠沉淀及在Sepharose 4B柱分离的方法从血液冷沉淀制剂中纯化。纯化的vWF固着在盖玻片上 ,在平行板液流室中进行细胞滚动研究 ,用相差电视显微镜观察。结果 表达GPⅠb Ⅸ Ⅴ复合物的CHO细胞能粘附于并滚动在固着的vWF表面 ,当用表达A15 6V突变的细胞进行试验时 ,这些细胞虽然能粘附于并滚动在固着的vWF表面 ,但是它们滚动的速度明显快于其野生型 ,这表明突变的GPⅠbα与vWF之间的受体配体键的解离速度受损 ,单克隆抗体AN5 1与突变的GPⅠbα结合明显减少 ,表明A15 6V突变产生了GPⅠbα氨基端配体结合区的构象改变。结论 突变致使A15 6V突变细胞与固着的vWF产生较快的解离速度。突变的多肽在GPⅠbα氨基端配体结合区发生构象改变。平行板液流室在评价GPⅠbα与vWF之间相互作用中是一种有用的工具
Objective To investigate the significance of the GP Ⅰ bα mutation (A15 6V) in the interaction between immortalized von Willebrand factor (vWF) and cells expressing mutations under fluid shear stress. Methods The mutations induced directly in GPIbα cDNA were cloned into the EcoRI site of the mammalian expression vector pDX, followed by transfection of the mutated cDNA into CHOβ IX cells. Human vWF was purified from blood cryoprecipitate formulation by precipitation of glycine and sodium chloride and separation on a Sepharose 4B column. Purified vWF was immobilized on coverslips, and cell rolling studies were performed in parallel plate flow chambers and phase contrast TV microscopy was used. As a result, CHO cells expressing the GPIb IX V complex adhered to and rolled on the surface of the immobilized vWF, and although these cells adhered to and rolled on the surface of the immobilized vWF when tested with cells expressing the A15 6V mutation They roll significantly faster than their wild type, indicating that the rate of dissociation of the acceptor ligand bond between the mutant GPIb [alpha] and vWF is impaired and the binding of the monoclonal antibody AN51 to the mutant GPIb [alpha] is significantly reduced, indicating that the A156V mutation Conformational changes in the GPIbα amino-terminal ligand binding region were generated. Conclusion Mutations caused rapid dissociation of A15 6V mutant cells and immobilized vWF. The mutant polypeptide conformational changes in the GPIbα amino-terminal ligand binding region. Parallel plate flow chambers are a useful tool in evaluating the interaction between GPIbα and vWF