Ambra/Beclin1介导的内源性神经干细胞自噬对脊髓损伤功能修复的作用及机制

Activation of endogenous neural stem cells (ENSC) to promote functional recovery of spinal cord injury (SCI) is an ideal strategy. After SCI, ENSC in transient increase following a substantial reduction may be the key to restrict the repair effect of injured spinal cord. The anti-apoptosis treatment failed to prevent the decrease of ENSC. Recent studies have shown that autophagy in disease of central nervous system can promote nerve cell death. Ambral combined with autophagy gatekeeper "Beclin1" mediated autophagy of embryonic nervous development. It was also observed that ENSC autophagy can be activated in the SCI in our previous study. Therefore we persumed that: Ambral binding with Beclin1 activation ENSC autophagy may be the main reason for the decrease in the number of adult injured spinal cord ENSC. In this study, transmission electron microscope was used to detect autophagic bodies.Immunofluorescence and Western blot detection of autophagy molecular LC3 and its influence on the survival of NSC were observed in rat spinal cord ischemic injury and neural stem cells (NSC) hypoxia and glucose deprivation models. siRNA modulating Ambral expression to inhibit autophagy was adopted to observe its effect on the survival of ENSC and repair of injured spinal cord.With this research,we hope it will reveal the molecular mechanism of ENSC autophagy ,so as to provide a new strategy and method for SCI treatment and functional rehabilitation of paraplegia.

激活内源性神经干细胞（ENSC）功能是促进脊髓损伤（SCI）功能修复的理想策略,SCI后ENSC短暂增加又大量减少可能是制约其修复效果的关键，抗凋亡等常规治疗未能阻止ENSC的减少。新近研究发现,自噬在中枢神经系统疾病中能促进神经细胞死亡, Ambral与自噬守门人“Beclin1”结合可介导发育胚胎神经的自噬，我们前期研究也观察到SCI能激活ENSC自噬，故此推测：Ambral与Beclin1结合激活ENSC自噬可能是成体损伤脊髓ENSC数量减少的主要原因。本项目拟用成鼠脊髓缺血损伤和神经干细胞（NSC）缺氧缺糖模型，透射电镜检测自噬小体，免疫荧光和Western Blot检测自噬分子LC3，观察体内外NSC自噬的发生及其存活率的变化, 同时用siRNA调控Ambral抑制自噬,观察其对ENSC存活率及对损伤脊髓功能修复的作用,揭示ENSC自噬分子机制,为SCI治疗和截瘫康复提供新策略。

激活内源性神经干细胞（ENSC）功能是促进脊髓损伤（SCI）功能修复的理想策略。SCI后ENSC短暂增加又大量减少可能是制约其修复效果的关键，抗凋亡等常规治疗未能阻止ENSC的减少。近年研究发现,自噬参与了中枢神经系统疾病的发生发展。自噬基因 Ambral与自噬守门人“Beclin1”结合可介导发育胚胎神经的自噬，我们前期研究也观察到SCI能激活ENSC自噬，故此推测：Ambral与Beclin1结合激活自噬可影响损伤脊髓ENSC数量及对神经功能。. 因此，本项目拟用神经干细胞（NSC）氧糖剥夺和成鼠脊髓缺血损伤模型，分别在离体和在体条件下观察NSC损伤后自噬的发生情况，以及通过Ambral与Beclin1调控自噬后对NSC存活和分化影响，同时观察了自噬对脊髓损伤后神经元的保护作用及对神经功能修复。. 首先我们在离体实验中构建NSCs氧糖剥夺模型，发现NSCs在氧糖剥夺损伤后会出现自噬分子如Beclin1、LC3B和P62等的表达，但持续的时间较短，NSCs死亡细胞数量多，分化能力差。通过Ambra1过表达腺病毒转染上调自噬分子Beclin1的表达，自噬强度增加、持续时间延长；使NSCs的存活率升高，生长状态较好，分化为神经元的比例也较高，初步证实自噬有利于损伤NSCs的存活和分化。. 其次在体实验中通过构建脊髓缺血损伤模型，我们发现脊髓中央管周围NSCs会出现自噬活动，但持续时间较短，1天后开始下降，7天时与正常对照组无显著差异。转染Ambra1过表达腺病毒后，自噬活动增强，自噬分子Beclin1、LC3B等的表达增加，3-7天时NSCs增殖细胞数量较对照组显著，提示自噬对NSCs存活有保护作用。进一步我们观察到缺血损伤后，脊髓前角神经元在3h左右有明显的自噬，随后自噬活动逐渐减弱，3天时接近对照组水平。利用自噬激动剂和拮抗剂干预后，激动剂组大鼠自噬的强度增加，7天时仍可观察到较明显的自噬现象，并且神经元存活数量较多，形态较好，其神经行为学和功能显著优于对照组。. 综上所述，本研究初步证实了脊髓缺血损伤后通过Ambra1激活自噬分子Beclin1对ENSC和神经元存活有较好保护作用，能显著改善神经行为和功能。因此，自噬有望成为神经系统损伤修复与康复治疗的新靶点。