TAT-LBD靶向介导的双靶点效应蛋白促进缺血性脑卒中神经功能修复及其机制研究

About three quarters of the patients who recovered from stroke and cerebral hemorrhage were obsessed by neurological functional deficit in varying degrees, which are mainly due to the weak regenerating and repairing functions of Central Nerve System (CNS). Thus, it is vital to protect and repair the remaining neurons. However, three inhibitory factors including NogoA, MAG and Omgp were introduced by injuried CNS, which could repress the functional recovery of the remaining neurons as well as the axon regeneration. Though blocking the NgR1 by NEP1-40 could promote the axonal regeneration and protect the neuron, it only has a short-term effect. In this project, our researching group is trying to block both the NgR1 and PriB receptor, and lead the way in producing new fusion proteins, TAT-LBD-NEP1-40 and TAT-LBD-PEP, which not only could block the NgR1 and PriB receptors at the same time, but also could pass the Brain blood barrier (BBB) and aggregate targeting the ischemia tissue. The effects of these two fusion proteins on remaining neurons functional recovery will be evaluated by both in vitro and in vivo experiments. Its molecular mechanism will be assessed based on NgR1/RhoA/ROCK, NgR1/Bcl2/Bax, PirB/POSH/ Shroom3/ROCK, PirB/SHP2/BDNF/TrkB signal pathway, in order to provide pharmacological theory for the development and application of specific effectors， and also this study will offer new theory and strategy for the neuroprotection of ischemic cerebral stroke.

缺血性脑卒中幸存患者往往伴有不同程度的神经功能障碍，主要原因之一是脑缺血损伤后NogoA等抑制因子通过NgR1和PirB受体途径不仅抑制轴突再生，还可加重神经元损伤，致使神经功能恢复困难。以往研究发现，单一干涉NgR1或PirB基因表达，仅能有限促进部分神经功能恢复，且难以向临床应用转化。我们前期及预实验发现，应用NEP1-40拮抗NgR1可在脑缺血损伤早期促进神经功能恢复，应用PEP阻断PirB可促进神经元存活和轴突生长。上述结果强烈提示：在蛋白质水平同时阻断NgR1和PirB受体途径将可显著改善神经功能缺失。因此，本课题拟制备可同时阻断NgR1和PirB功能且可靶向集聚在缺血区的TAT-LBD-NEP1-40和TAT-LBD-PEP的融合蛋白，利用OGD和MCAO等模型，综合研究两种功能蛋白联合应用对脑缺血损伤的治疗作用并探讨分子机制，为脑缺血后的神经功能恢复及该蛋白应用提供理论依据。

本项目严格按照计划书的进度、内容和目标要求执行。实验采用可同时阻断 NgR1 和 PirB受体功能的 TAT-LBD-NEP1-40 和 TAT-LBD-PEP（PirBex） 的2 种融合蛋白，利用 OGD 和MCAO 等模型，完成了对脑缺血损伤的治疗作用及相关分子机制。主要结果为：①成功获得TAT-LBD-NEP1-40和TAT-LBD-PirBex融合蛋白；②应用免疫组化方法，发现2种融合蛋白能够穿过血脑屏障和细胞膜，并具有脑缺血区聚集功能；③证实2种融合蛋白与NgR1 和 PirB受体具良好亲和力；④应用细胞培养OGD 模型，研究证实二种融合蛋白具有促进轴突生长、侧枝形成及促进神经元存活的作用，并有剂量依赖性；⑤以MCAO模型，用MRI和TTC染色方法，证实二种融合蛋白能减少脑梗死容积，改善MCAO后动物肢体力量，运动，协调和平衡等功能，提高神经行为学评分，促进神经功能恢复的作用；⑥应用BDA皮层定位注射和免疫组化方法，证实二种融合蛋白能够促进MCAO后动物皮质脊髓束神经元的轴突和树突的生长及分支的发生，提高其重塑能力；⑦应用免疫组化方法，证实二种融合蛋白能够加强MCAO后动物大脑皮层半暗带内神经元NF200、MAP2、SYN、HOSH、RhoA和GAP43的表达；⑧以原代培养皮层神经元，用共聚焦活细胞工作站检测方法，证实融合蛋白能够明显促进OGD后神经元轴突及其分支生长；⑨电生理学方法 证实融合蛋白可以提高离体脑片OGD后神经元sEPSC的振幅和频率，促进突触可塑性的恢复。研究结果提示2 种融合蛋白具有同时阻断NgR1和PirB两种受体的功能，能够有效的促进缺血/缺氧损伤后残存神经元功能修复和促进轴突生长的作用，为NgR1和PirB 作为联合干预靶点提高CNS神经功能修复提供一个新的理论依据。. 研究成果在国内外期刊正式发表论著12篇，其中SCI论文5篇，国内核心期刊7篇。 获专利10项，其中国家发明专利5项，国际发明专利5项。获省部级科技进步二等奖2项，陕西省优秀博士论文和自然科学优秀论文各1篇。人才培养7名，其中博士后2名，博士4名，硕士1名。按期完成全部研究内容。