二氧化硅纳米颗粒诱导多巴胺能神经元α-突触核蛋白聚集及其机制研究

As nanotechnology and materials science have rapidly emerging, engineered nanomaterials have been widely applied and as a result, nanotoxicology research is now gaining a great deal of attention. In the previous work, we have found that silica nanoparticles (SiO2-NPs) could enter into brain and especially accumulated in the striatum via intranasal instillation. The deposition of SiO2-NPs in the striatum could induce inflammatory response and oxidative damage. Meanwhile, oxidative stress is considered as an critical factor in the aggregation of α-synuclein which is a crucial pathopoiesia protein for Parkinson's disease. On the basis of literature and previous work, we present a hypothesis that nanoparticles could induce α-synuclein aggregation in dopaminergic neurons through oxidative stress, and the possible mechanism involved in the impair of protein degradation pathways. In the present study, we investigate the effect of SiO2-NPs on the α-synuclein aggregation at in vivo, cell and molecular level and intended to clarify the mechanisms underlying the ubiquitin-proteasome system and autophagy-lysosom pathway. Based on these results, possible risk of SiO2-NPs for Parkinson's disease is well explored at different aspects. Our study lay a foundation for the research on the neurotoxicity and application risk of nanoparticles, and has an important significance for the development of nanotechnology.

纳米材料的生物安全性是当今纳米科技发展中亟待解决的核心科学问题之一。本项目组前期研究发现，二氧化硅纳米颗粒（SiO2-NPs）在脑组织中主要分布于纹状体和海马，并引发纹状体的氧化损伤。而氧化应激为帕金森病致病关键蛋白α-突触核蛋白聚集的重要途径。结合文献及既往工作基础，我们推测：纳米颗粒可通过氧化应激诱导多巴胺能神经元α-突触核蛋白聚集，且细胞蛋白降解通路的功能障碍在该损伤途径中发挥关键作用。本项目以SiO2-NPs对多巴胺能神经元的损伤为切入点，结合现代分子生物学和免疫学技术，从整体、细胞和分子三个水平研究SiO2-NPs对α-突触核蛋白聚集的影响及作用途径，揭示泛素-蛋白酶体系统和自噬在SiO2-NPs诱导α-突触核蛋白聚集中的作用及机制，在不同层面上，探讨纳米颗粒诱发帕金森病的潜能，为纳米颗粒的神经毒性效应研究和规避纳米颗粒的应用风险奠定基础，对促进纳米科技发展具有积极和重要意义。

近几年，纳米颗粒被认为与神经退行性疾病如阿兹海默症和帕金森病的发生有密切关系，因此纳米颗粒的神经毒性效应已成为目前的研究热点。本研究在前期研究的工作基础上，通过建立SiO2-NPs与多巴胺能神经元的共培养模型，研究SiO2-NPs对帕金森病的致病关键蛋白α-Synuclein聚集的影响及作用机制。我们采用western blot及免疫荧光技术检测PC12细胞内α-Synuclein的表达水平，同时将NAC和SiO2-NPs混合物一起处理PC12细胞，来比较添加NAC和未添加NAC的SiO2-NPs对PC12细胞α-Synuclein表达的影响。采用荧光酶标仪及western blot技术检测20S蛋白酶体活性及泛素蛋白酶体系统关键蛋白表达。通过透射电镜检测及western blot技术检测细胞自噬行为及自噬分子机制。结果发现SiO2-NPs可诱导α-synuclein表达上调及其聚集体的形成，该效应与氧化应激有关。进一步的分子机制研究表明，SiO2-NPs可损伤 20S 蛋白酶体活性及UPS的关键蛋白表达，同时可通过PI3K-AKT-mTOR信号通路诱导PC12细胞发生自噬现象，由于本实验中α-synuclein聚集形式可能主要是泛素化状态，所以SiO2-NPs诱导α-synuclein聚集效应主要与UPS的损伤有关。本研究提示，SiO2-NPs暴露具有潜在神经毒性风险，同时具有诱发帕金森病的潜能。