【作者】 王宁；

【导师】 李静；

【作者基本信息】 武汉理工大学 ， 生物医学工程， 2023， 硕士

【摘要】 在阿尔兹海默症（AD）病理研究中,淀粉样蛋白Aβ级联病理机制具有重要位置。病理状态下,Aβ从可溶性转变到不溶性的富含β-层状折叠的纤维化聚合物,该聚合物具有引发碱性神经元损伤、神经细胞凋亡、氧化应激等毒性特性。大多数研究都关注Aβ的结构和毒性作用,但是细胞膜作为错误折叠蛋白的最直接靶点,它们之间的相互作用会影响细胞膜的生物学特性,研究Aβ与细胞膜的相互作用有利于深入了解淀粉样蛋白的致病机制。因为纳米材料的独特的物理和化学特性,以及它们的尺寸、结构和配体的可修饰性、可控释药物等。本论文在探索细胞膜成分与Aβ的相互作用机制的基础上,进一步探索氧化铁（Fe₃O₄）纳米材料对Aβ的调控作用,为AD的发病机制及药物筛选提供基础。本文主要研究内容如下:（1）构建了三种不同磷脂模型,即二油酰基卵磷脂（DOPC）和1,2-二棕榈酰-sn-甘油-3-磷酰胆碱（DPPC）以及1,2-二油酰基-sn-甘油-3-磷酰-rac-（1-甘油）钠盐（DOPG）。通过磷脂囊泡表征,发现DPPC和DOPC为中性,DOPG带负电荷,DOPC、DOPG形成松散的膜结构,DPPC形成紧密堆积的结构。合成的磷脂囊泡粒径范围在100～200 nm的范围内,三种磷脂囊泡的CD光谱相似,信号强度有区别。（2）探究三种磷脂膜对Aβ纤维化的影响。利用Th T荧光动力学实验、原子力显微镜（AFM）、动态光散射（DLS）、圆二色光谱仪（CD）等表征手段发现三种磷脂囊泡都会对20μmol·L^-1 Aβ₄₀聚集有抑制作用,但机制不同,20μmol·L^-1Aβ₄₀在DOPC脂质膜上会形成环状结构的纤维;在中性磷脂膜上（DOPC、DPPC）形成很短的纤维（小分子量聚集体）,并且会在破坏磷脂膜的完整性,在磷脂膜上形成孔洞;20μmol·L^-1 Aβ₄₀在带负电荷的磷脂膜上（DOPG）形成长纤维（大分子量聚集体）,但相较于不在磷脂膜上的,纤维长度变短,可见DOPG磷脂膜影响了Aβ₄₀的聚集。（3）探索了Fe₃O₄纳米材料对Aβ纤维化的影响。使用水热法合成了PEG-Fe₃O₄,通过透射电镜（TEM）、傅里叶变换红外光谱（FT-IR）、X-射线光电子能谱（XPS）等测试方法对其进行表征,通过酶活性检测,结果表明PEG-Fe₃O₄ NPs具有较高的SOD活性和CAT活性。使用Th T荧光动力学、TEM、DLS和CD光谱进一步证实了低浓度的纳米级的PEG-Fe₃O₄对Aβ40纤维化动力学,聚集和构象变化有明显的抑制作用。并通过细胞实验进一步验证了低浓度的PEG-Fe₃O₄对Aβ₄₀纤维化的抑制作用。这项研究揭示了Aβ与仿生细胞膜之间的相互作用,为深入理解Aβ肽诱导细胞毒性的机制提供了见解。此外这项工作也研究了Fe₃O₄纳米酶对淀粉样蛋白的调控作用,为四氧化三铁纳米酶用于治疗神经性疾病提供了数据支撑。更多还原

【Abstract】 In the study of Alzheimer’s disease（AD）,the pathological mechanism of the amyloid Aβcascade plays an important role.In the pathological state,Aβchanges from a soluble state to an insoluble fibrotic polymer rich inβ-layered folding,which has toxic properties such as alkaline neuron injury,nerve cell apoptosis and oxidative stress.Most studies on Aβfocus on conformational transformation and toxic effects.However,as the most direct target of misfolded proteins,the interaction between Aβand the cell membrane will affect the biophysical properties of the cell membrane.The study of the interaction between Aβand the cell membrane is beneficial to further understand the pathogenic mechanism of amyloid.Because of their unique physical and chemical properties,size,structure and strong modifiability of ligands,nanomaterials have the advantage of controllable drug loading.Based on the exploration of the interaction mechanism between cell membrane components and Aβ,this thesis further explores the regulatory effect of iron oxide（Fe₃O₄）nanomaterials on Aβto provide a basis for the pathogenesis of AD and drug screening.The main research of this paper is as follows:（1）Three different phospholipid models were constructed,Dioleyl lecithin（DOPC）and 1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine（DPPC）and 1,2-dioleoyl-sn-glycerol-3-phosphoryl-rac-（1-glycerol）sodium salt（DOPG）.By characterization of phospholipid vesicles,it was found that DPPC and DOPC were neutral,DOPG was negatively charged,DOPC and DOPG formed a loose membrane structure and DPPC formed a tightly packed structure.The size distribution of the synthesized phospholipid vesicles was uniform,ranging from100 nm to 200 nm,The CD spectra of the three phospholipid vesicles were similar and the signal intensity was different.（2）To explore the effects of three phospholipid membranes on Aβfibrosis.Through Th T fluorescence kinetic assay,atomic force microscopy（AFM）,dynamic light scattering（DLS）,circular dichroism spectrometer（CD）and other characterization methods,it was found that all three kinds of phospholipid vesicles inhibited the aggregation of 20μmol·L^-1 Aβ₄₀,but the mechanism was different.20μmol·L^-1 Aβ₄₀ formed ring structure fibers on the DOPC lipid membrane.Short fibers（small molecular weight aggregates）were formed on neutral phospholipid membrane（DOPC,DPPC）at 20μmol·L^-1 Aβ₄₀,and the integrity of the phospholipid membrane was destroyed,and holes were formed on the phospholipid membrane.20μmol·L^-1 Aβ₄₀ formed long fibers（large molecular weight aggregates）on the negatively charged phospholipid membrane（DOPG）,but the fiber length was shorter than that on the non-phospholipid membrane,indicating that the aggregation of Aβ₄₀ was affected by the DOPG phospholipid membrane.（3）The effect of Fe₃O₄ nanomaterials on Aβfibrosis was explored.PEG-Fe₃O₄ was synthesized by hydrothermal method and characterized by transmission electron microscopy（TEM）,Fourier transforms infrared spectroscopy（FT-IR）,and X-ray photoelectron spectroscopy（XPS）.The results showed that PEG-Fe₃O₄ NPs had high SOD and CAT activities by enzyme activity detection.The inhibitory effect of low concentration of nanoscale PEG-Fe₃O₄ on the kinetics,aggregation and conformational transition of Aβ₄₀ fibrosis was further confirmed using Th T fluorescence kinetics,TEM,DLS and CD spectroscopy.The inhibitory effect of low concentration of PEG-Fe₃O₄ on Aβ₄₀ fibrosis was further verified by cellular experiments.This study reveals the interaction between Aβand biomimetic cell membranes,providing insight into the mechanism of Aβpeptide induced cytotoxicity.In addition,this work also investigated the regulatory effect of Fe₃O₄nanomases on amyloid protein,providing data support for the application of ferric oxide nanomases in the treatment of neurological diseases.更多还原