淀粉样多肽自组装过程及其对磷脂膜损伤机制的体外重现及理论模拟

The main mission of this project is to construct a self-developed matrix system as an in vitro template to simulate the in vivo reactions among different aggregates of β-amyloid peptide (Aβ) and neuron cell that may take place in human brain. Our previous experimental results convinced that introducing Ferrocenyl group in to certain peptides did not significantly change the self-assemble behaviors of the original peptides. We hereby use ferrocenyl derived peptides as the molecular probes to characterize the structural changes of Aβ during it's self-assembly process. The investigation of the penetration behavior of Aβ aggregates into supported lipid bilayer membranes surfaces is the key to mimic the interactions among all the aggregates of Aβ and neuron cells. In reviews all the interactions among Aβ and lipid membrane, the fastest aggregating speed was achieved at acidic conditions, while main products are fibirlls. The second mission of this project is to prove our original proposes of building up a new protocol for quick and High-throughput drug screening of Alzheimer Disease.It need to be pointed out that our AFM morphology images confirmed that Aβ spherical oligomers could create irreversible "holes" into supported bilayer while Aβ fibers were also able to embed themselves into the supported lipid membranes. However, when we removed Aβ fiber from the membrane with AFM tips, the deformed lipid membranes were able to recover to the initial states. This discovery yields the mechanical explanation that why the toxicity of Aβ oligomers is much higher than Aβ fibril. Consequently, the drug screen principle on potential candidates for Alzheimer Disease needs to be renewed. We are going to select the fibirllization inductors as their objective drugs rather than the β-sheet breakers. We here plan to combine two well-established techniques: atomic force microscopy (AFM) and scanning electrochemical microscopy (SEM) to monitor the efficiencies of potential drugs in encouraging the fibrillation of Aβ monomers，respectively. The results that obtained from our method were in good agreement with the cell viability tests.

β-淀粉样多肽(Aβ)分子的自组装失控被认为是最有可能导致阿尔茨海默病的因素之一。本课题得益于"与Aβ特定片段具有相同氨基酸序列的二茂铁多肽具有和Aβ自身一致的自组装行为"这一原创发现，计划使用各种二茂铁短肽体系来体外重现Aβ的自组装-解组装过程。核心的研究思路是：构筑一个和人体大脑环境近似的体系，改进现有界面化学的分析测试手段，现场观测具有不同氨基酸序列的二茂铁短肽自组装-解组装的动力学行为，鉴定不同二茂铁短肽自组装基元在其各种类型聚集体中堆积方式的异同。结合理论计算方法的改进，归纳总结二茂铁短肽不同类型聚集体相互之间转化过程中规律性的信息。本课题的成功实施，将为复杂生物体系的研究提供一种实时、微观的原位分析方法；并由此提出在分子、微区、界面和溶液等不同层面观测细胞膜的病变行为及药物相互作用的动力学过程的新建议；为相关技术在新药开发等领域的潜在应用提供理论依据。

在本项目基金的资助下，工作按计划进行，取得了一系列的进展，研究成果主要包括以下几个内容：1. 钙离子诱导的多肽磷脂相互作用。课题组按计划合成了一系列的短肽，从头计算（DFT），得到了多个小分子多肽的单体、二聚体以及寡聚体的最低能态构型，预见到了氢键的生成并计算出了可能的生成熵值。我们的光谱表征结果证实了氢键的存在。实验结果表明， 这些潜在的氢键，在多肽与磷脂膜的相互作用过程中至关重要。此外，我们的研究还发现，钙离子在多肽的聚集过程中，也起到了非常关键的作用。我们推测钙离子在带负电的磷脂表面形成了对多肽的结合位点，从而使得多肽接近磷脂膜过程中的能垒大大降低。实验结果进一步表明，寡聚体通过与镶嵌进入磷脂膜内部，形成孔洞结构造成细胞凋亡。但是，与此前文献报道不同的是，我们在实验中观测到，磷脂膜本身的完整性并没有收到破坏。2. 能够加快淀粉样多肽纤维的天然产物的筛选，在成果1的基础上我们挑选了一系列的天然产物，其具有加快淀粉样多肽聚集的效果，实验结果显示，其中大黄酸分子能够形成凝胶，实现在淀粉样多肽存在下对神经细胞的生长进行保护。3. 短肽金属凝胶的设计制备及诱导神经细胞实验分化的研究。在研究金属离子对短肽与磷脂膜相互作用中，我们首次发现一些特定的短肽可以通过与金属离子相互作用，形成水凝胶，进而保护磷脂膜不受淀粉样多肽的损伤。细胞实验的结果表明，有一类金属短肽凝胶可以促进神经细胞的突触生长，分化，交联。这一突破性的发现，为我们研究部分恢复神经退行性疾病患者记忆的研究提供了体外实验证据。