模型膜实验方法在β淀粉样蛋白与细胞膜间相互作用中的应用与改进

Application and Improvement of Model Membrane Experimental Method in the Interaction Between Amyloid β Peptide and Cell Membrane

淀粉样蛋白(amyloidβpeptide,Aβ)与细胞膜间的相互作用很可能是阿尔茨海默症病(Alzheimer disease,AD)重要的风险因素。模型膜研究方法在该领域的应用和更新持续至今,但仍存在一些问题有待解决,例如,Aβ插膜后聚集状态与Aβ融合到脂质体膜聚集状态的差异,Aβ插膜后形成微通道的时间及与磷脂成分的关系等。本文试图解析这两个问题,同时,系统地总结出常用的和更新的模型膜研究方法,这些方法包括单层膜插膜及电镜样品的制备,脂质体制备方法的改进,脂质体膜上Aβ42经过高盐及酸清洗后的Western印迹检测,ANTS?DPX研究脂质体泄漏等。研究结果显示:(1)胞外及膜内Aβ42单体与脂质体膜作用后的聚集状态存在差异,Aβ42单体插膜后更容易聚集成纤维,而膜内融合的Aβ42呈现寡聚体形式;(2)Sepharose CL?4B柱过滤比微型挤出器制备的脂质体更加均一分散;(3)Aβ42在膜上形成微通道很可能是一个缓慢的过程,且与脂质体的磷脂种类相关。这些方法为Aβ42与细胞膜的相互作用提供了实用的研究手段,同时也为其他膜蛋白质与细胞膜的相互作用提供了可以借鉴的办法。研究结果使β淀粉样蛋白代谢过程更加清晰。

The interaction between amyloid p peptide(Aβ)and cell membrane is likely to be an important risk factor for Alzheimer’s disease(AD).The application and update of model membrane research methods in this field have continued up to now.However,there are still some problems to be solved,such as the differences between the aggregation of Aβ after insertion and the fusion of Aβ into the aggregation of liposome membrane,and the time of formation of microchannels after Aβ insertion and the relationship with phospholipid composition,etc.In this paper,we attempt to resolve these two problems.Meanwhile,we systematically summarize the common and updated research methods of model membranes.These methods include monolayer insertion and preparation of electron microscopic samples,improvement of preparation methods of liposomes,Western blot detection of Aβ42 on liposome membranes after high salt and acid cleaning,and ANTS-DPX study of liposome leakage.The results showed that(1)there were differences in aggregation of Aβ42 monomers and liposome membranes in extracellular and intramembranous media,and Aβ42 monomers were more likely to aggregate into fibre after insertion,while the fused Aβ42 showed oligomer form;(2)Sepharose CL-4β column filtration was more uniform than that of liposomes prepared by micro-extruder;(3)the formation of microchannels on liposomes is likely to be a slow process and is related to the types of phospholipids in liposomes.These methods provide a practical application to study the interaction between Aβ42 and cell membrane,and also provide a reference for the interaction between other membrane proteins and cell membrane.The results help further understanding the metabolic process of p-amyloid protein.However,the process of microchannels formation and clear conformation of p-amyloid protein on model membrane remain to be clarified.