心室辅助装置引发血管性血友病因子损伤的检测方法研究

Study on the detection of mechanical damage to von Willebrand factor by ventricular assist device

目的由于血液成分血管性血友病因子(von Willebrand factor,vWF)的机械损伤现象发现和研究较晚,至今仍然定义模糊且缺乏相关的评价标准,非常不利于心室辅助装置(ventricular assist device,VAD)的创新设计与发展。本文通过研究分析,提出检测vWF受高剪切应力损伤的实验方法。方法人血作为基础血样,离心式血泵BPX-80剪切前后的猪血作为测试血样,通过免疫印迹法,将电泳分离的vWF转移到膜上,然后用特异性抗体检测膜上vWF多聚体的分子量分布情况,根据vWF分子量的灰度值比值,定量分析vWF多聚体的机械损伤,整个实验过程包括制胶、电泳、转印、免疫反应和显色5部分。结果得到条带清晰完整且容易区分的vWF分子量分布图。其中健康人血vWF高分子量、中分子量、低分子量区域的灰度值比值与文献中健康人血vWF的实际比值相一致。BPX-80血泵剪切猪血中高分子量vWF与未经剪切的猪血高分子量vWF灰度值的比值随时间呈下降趋势,表明BPX-80血泵剪切对猪血中vWF的降解情况。结论成功设计并建立了vWF多聚体分析实验方法,为此后制定vWF机械损伤的标准化体外评价方案提供重要参考。

Objective Due to the late discovery and study of the mechanical injury phenomenon of blood component von Willebrand factor(vWF),it is still poorly defined and lacks relevant evaluation criteria,which is not conducive to the innovative design and development of ventricular assist devices.A method is proposed to evaluate the damage of the vWF induced by high shear stress though research and analysis.Methods Human blood was used as the basic blood sample for the test,pig blood samples sheared by centrifugal blood pump BPX-80 were used as the test samples.We transferd the vWF separated by electrophoresis and Western blot to PVDF membrane,then used specific antibody detection of vWF distribution,quantitative analyzed mechanical damage of vWF according to the gray value ratio of molecular weight.The whole experiment process included glue making,electrophoresis,transfer printing,immune reaction and color developing.Results The vWF molecular weight distribution map with clear and complete bands was obtained.The gray value ratio of high,medium and low molecular weight vWF in healthy human blood was consistent with the ratio of vWF in healthy human blood in literature.The ratio of high molecular weight vWF gray value in BPX-80 blood pump decreased with time,indicating the damage of vWF by BPX-80 blood pump.Conclusions The experimental method of vWF polymer analysis was successfully designed and established,providing an important reference for the further development of standardized in vitro evaluation scheme of mechanical damage of vWF.