Shh通路介导运动训练促进外源性神经干细胞在脑梗死大鼠纹状体增殖分化的机制研究

Neurogenesis is significant for the recovery of neurological functions after stroke. For endogenous neurogenesis is limited, neural stem cells (NSCs) transplantation may pave a new way for rehabilitation of stroke，but the molecular mechanisms behind the proliferation and differentiation of NSCs in vitro has not yet been elucidated. In our previous study, we induced NSCs use induced pluripotent stem cells (iPSCs) from human urine epithelial cells in vivo. When grafted into the striatum of cerebral ischemia rats, the NSCs survived but only a very few of them differentiated into GABAergic neurons. We also found exercise training promoted the proliferation of endogenous NSCs after stroke, increased the number of striatal neurons around the infarction area and the expression of sonic hedgehog (Shh) simultaneously. The related research suggests that NSCs from ESCs can be induced to GABAergic neuron by Shh in vitro. Therefore, we propose that exercise training after stroke may promote the NSCs generated from human urine epithelial cells proliferate and differentiate into GABAergic neurons by Shh up-regulation. Male SD rats subjected to transient focal cerebral ischemia by middle cerebral artery occlusion are used in this experiment. All animals in exercise group submitted to the running wheel exercise were placed into a programmable, motorized wheel apparatus. Neurologic function is tested by mNss score. The proliferation and differentiation of NSCs are detected by CLARITY and immunofluorescence staining. The role of Shh/Smo/Gli1 pathway are detected by immunofluorescence staining, RT-PCR and western blot. The results may contribute to further understand the proliferation and differentiation mechanisms of NSCs in vitro and optimize rehabilitative therapy of stoke in clinical medicine.

神经再生是脑卒中后神经功能恢复的重要机制，但内源性神经干细胞（NSCs）作用有限，干细胞移植可为神经再生提供可靠的细胞来源。移植的外源性干细胞如何在脑内增殖分化成有功能的神经元是决定修复疗效的关键。GABA能神经元是脑卒中最常见部位—基底节的主要神经元，Shh通路促进NSCs向GABA能神经元分化。我们发现，运动训练能上调Shh及促进脑梗死后内源性NSCs的增殖分化。预实验显示，在脑梗死大鼠纹状体移植人尿源iPSCs诱导的NSCs可部分分化成GABA能神经元。我们假设，运动训练可通过激活Shh通路促进外源性NSCs增殖分化成GABA能神经元并改善神经功能。本研究拟构建大鼠tMCAO运动训练模型，采用脑组织透明化成像技术、RNAi等方法，检测外源性NSCs在纹状体的增殖分化及Shh通路在其中的作用，阐明脑梗死后运动训练对外源性NSCs增殖分化的作用及机制，为干细胞移植治疗脑卒中提供实验证据。

神经再生是脑卒中后神经功能恢复的重要机制，神经干细胞（neural stem cells，NSCs）是神经再生的主要来源，但内源性NSCs作用有限，干细胞移植可为神经再生提供可靠的细胞来源。然而，移植的外源性干细胞如何在脑内增殖分化成有功能的神经元是决定修复疗效的关键。本项目在前期研究的基础上，进一步研究脑梗死后运动训练是否能促进脑梗死后移植的外源性NSCs的增殖，并促进其向神经元分化，进而更好地改善脑梗死大鼠的神经功能，并探讨Shh（sonic hedgehog）信号通路在其中的作用。. 本研究通过构建大鼠大脑中动脉栓塞再灌注（transient middle cerebral artery occlusion，tMCAO）模型，采用运动训练联合NSCs移植，并采用Brdu注射、Tunel染色、脑组织免疫荧光等方法，观察运动训练对移植的NSCs增殖、分化的影响，分析Shh信号通路的作用，并进一步采用ELISA、RNA-seq方法，探讨运动训练促进移植的NSCs增殖分化的机制。研究发现：运动训练联合NSCs移植较单独NSCs移植能更好地减小脑梗死大鼠的梗死体积，并促进神经功能恢复；进一步研究发现，运动训练促进移植的NSCs向神经元分化，增加移植细胞中神经元的比例；运动训练促进移植的NSCs向成熟的神经元分化，促进分化后的神经元突触形成，增加神经元活动，并融入宿主神经回路；其潜在机制可能与运动促进脑梗死周围区微血管的生长和增强神经纤维再生，同时运动训练联合NSCs移植，通过增加神经营养因子及生长因子的表达，调节梗死周围区神经元生存的微环境有关。. 本项目的研究结果与预期实验结果相符，达到了本项目的研究目的，为将来临床应用干细胞移植治疗脑卒中提供了实验证据，也为探索脑卒中后康复联合治疗策略提供了新的思路。