狂犬病毒糖蛋白负载siRNA-neogenin治疗急性运动轴索型神经病的实验研究

Acute motor axonal neuropathy (AMAN) is the most frequent subtype of Guillain-Barrés syndrome in China; and the anti-GD1a antibodies (Abs) serve as the pathogenic factor in AMAN. Recent studies have demonstrated that anti-GD1a Abs can promote the expression of RhoA to inhibit the regeneration of the impaired axon; however, its definite mechanism remains to be elucidated. Our previous study has demonstrated that neogenin, the receptor of axon guidance molecules may be the key factor to mediate the mechanism of anti-GD1a inhibiting the regeneration of axon in AMAN. In this study, we are aimed to construct the dominant negative mutant vector of neogenin and the Luciferase reporter gene vector of RhoA promotor, both of which will be transfected into the motor neuron of the mice spinal cord anterior horn in vitro to explore the molecular mechanism of neogenin inhibiting the axon regeneration; meanwhile, with the neurotropic characteristic, rabies viral glycoprotein together with the RNA interference will be used to mediate the target siRNA to silence the expression of neogenin in AMAN mice. According to the clinical symptoms, pathological examination and the expression of of immune molecules, we will evaluate the effects of neogenin deficiency on the axon regeneration of AMAN mice to finnaly confirm whether neogenin can be the new target for the clinical therapy of AMAN.

急性运动轴索型神经病（AMAN）是吉兰-巴雷综合征在我国最常见的、预后差的发病类型，其致病性抗体为抗GD1a抗体。最新研究表明，抗GD1a抗体可通过上调RhoA的表达来抑制AMAN中的轴索再生，然而其具体机制仍不明确。本课题组在前期的预实验中发现：轴索导向因子受体-neogenin可能是介导抗GD1a抗体抑制轴索再生机制中的关键分子。本项目旨在构建neogenin显性失活突变体和RhoA启动子荧光素酶表达载体，转染体外培养的小鼠脊髓前角运动神经元，探讨neogenin介导轴索再生抑制作用的具体机制；同时利用狂犬病毒糖蛋白嗜神经的特点，结合RNA干扰技术，靶向性沉默AMAN小鼠neogenin的表达，从临床、病理及免疫分子水平充分评估neogenin缺失对AMAN小鼠轴索再生的影响，最终明确neogenin能否成为临床治疗AMAN的新靶点。

急性运动轴索型神经病（AMAN）是吉兰-巴雷综合征在我国最常见的、预后差的发病类型，其根本的原因在于损伤后的轴索难以再生。本项目中，课题组利用小鼠脊髓前角运动神经元建立AMAN的细胞模型，发现Neogenin在AMAN细胞模型中的表达显著增高，利用Neogenin-siRNA抑制其表达后可显著促进AMAN运动神经元的轴索生长并增加生长锥的数目，该结果提示Neogenin可能是影响AMAN中轴索难以再生的关键分子，进一步的荧光素酶报告基因策略实验表明Neogenin可能通过上调RhoA的表达而抑制AMAN运动神经元的轴索再生。为了探讨Neogenin在临床治疗AMAN中的潜能，课题组利用GD3s-/-小鼠建立AMAN动物模型，利用狂犬病毒糖蛋白偶联Neogenin-siRNA并注入AMAN小鼠，通过临床评分、电生理和神经病理评估Neogenin缺失对AMAN模型的影响，我们的结果表明：狂犬病毒糖蛋白可有效的介导运动神经特异性的RNA干扰，Neogenin缺失可显著促进AMAN小鼠的轴索再生和临床症状改善。Neogenin有望成为临床治疗AMAN的新靶点，本项目的开展为AMAN的治疗提供了新的策略。