基于石墨相氮化碳纳米片的双基因协同输送系统用于小鼠胚胎神经干细胞定向诱导分化及脑内移植治疗帕金森病

Parkinson's disease (PD) is the second most common neurodegenerative disease. For the moment, neither drug therapy nor surgery can cure this disease fundamentally. Neural stem cells(NSCs) transplantation become a new view for PD, because lossing of the dopaminergic neurons(DA) in substantia nigra pars compacta, which is a pathological marker of PD. In this study, a g-C3N4 nanosheets with water solubility, positive charge and strong fluorescence is prepared in our previous work. and then the specific photoelectric properties and high specific surface area of g-C3N4 nanoplatelets are fully utilized to construct a dual-gene siSOX9/pNurr1 co-delivery system to achieve dual transfection of siRNA and plasmid DNA simultaneously. g-C3N4 nanosheets are used to co-deliver siSOX9 and pNurr1 into the embryoinc mouse NSCs, which enhance the directional differentiation dopaminergic neuron of NSCs, and further treatment of MPTP-injured mouse model of PD by stereotactic transplantation in the brain. The project will be plan to conduct in-depth systematic exploration from the following three aspects: the effect and mechanism of g-C3N4 nanosheets on the survival, proliferation and differentiation of mouse embryonic NSCs under light stimulation; the performance and mechanism of pNurr1-siSOX9-g-C3N4 system that induce directional differentiation into DA neurons of mouse embryonic NSCs under light stimulation; the therapeutic potential of brain transplantation in mouse model with the dual gene co-delivery system constructed by g-C3N4 nanosheets.

帕金森病（PD）是第二种常见的神经退行性疾病，目前无论是药物治疗还是外科手术，都不能从根本上治愈疾病。由于黑质致密部多巴胺能（DA）神经元的损失是PD的病理学标志，NSCs移植治疗为治愈PD带来新曙光。本项目拟制备水溶性、正电荷、强荧光的g-C3N4纳米片，在前期工作基础上，充分利用g-C3N4纳米片特异的光电性能及高比表面积，构建双基因siSOX9/pNurr1协同输送系统，实现siRNA和质粒DNA同时转染，级联式诱导小鼠胚胎NSCs定向分化成DA能神经元，并进一步通过脑内立体定位移植治疗MPTP损伤的小鼠PD模型。项目拟从以下三方面进行深入系统地探究：g-C3N4在光照条件下对小鼠胚胎NSCs的增殖、分化的作用及机制；pNurr1-siSOX9-g-C3N4在光照条件下诱导小鼠胚胎NSCs定向分化成DA神经元的性能及机制；g-C3N4构建的双基因协同输送系统应用于小鼠PD模型治疗潜能

通过移植神经干细胞（neural stem cells，NSCs）并促进NSCs增殖、诱导其定向分化为多巴胺能（dopaminergic neuron，DA）神经元，可以替代部分丢失的DA神经元的功能，有望治愈帕金森病（Parkinson’s disease，PD）。本项目在前期工作基础上，已制备了大量水溶性、正电荷、强荧光的g-C3N4纳米片。首先，探索g-C3N4纳米片对NSCs存活、增殖、分化的作用，通过Calcein-AM/PI染色与EdU染色证明了高浓度的g-C3N4纳米片（120 μg/mL）基本没有细胞毒性，并且90 μg/mL g-C3N4纳米片对NSCs的增殖能力有着明显的促进作用，通过GFAP与MAP-2 的免疫荧光染色结果证明了g-C3N4纳米片对NSCs向神经元分化具有正向的影响。同时，通过实验观察到，g-C3N4纳米片具有较好的基因负载率，且易被细胞摄取。其次，评估g-C3N4纳米负载双基因（pNurr1/siSOX9）后诱导小鼠胚胎NSCs定向分化成DA神经元的情况，通过RT-qPCR、Western Blotting实验证明了g-C3N4纳米片负载siSOX9、pNurr1、pFoxa2基因进入NSCs，对NSCs向神经元分化均有正向的影响，并且g-C3N4-siSOX9/pNurr1纳米复合物能够诱导NSCs向TH阳性神经元（DA神经元）分化。因此，最后选取g-C3N4-siSOX9/pNurr1纳米复合物用于探究小鼠PD模型脑内移植治疗的成效，采用6-羟基多巴胺（6-OHDA）成功构建PD小鼠模型，注射内化g-C3N4-siSOX9/pNurr1纳米复合物的小鼠胚胎NSCs，通过行为学实验和Western Blotting证明了内化g-C3N4-siSOX9/pNurr1纳米复合物的NSCs对PD小鼠有一定的治疗作用。这些工作为小鼠胚胎神经干细胞协同纳米材料脑内移植治疗帕金森病提供新策略。以本项目为基础，已发表SCI论文2篇，北大核心期刊论文6篇，指导10名硕士研究生毕业，指导3名硕士研究生获得广西研究生教育创新计划项目（区级），参加国内学术会议交流12次，与国内多个实验室建立稳定合作关系。