MiR-124调控脊髓损伤反应性星形胶质细胞分化为神经元的作用及机制研究

Reactive astrocytes (RACs), emerging after spinal cord injury (SCI), possess some neural stem cells characters. However, RACs don’t differentiate into neurons, but form glial scar and inhibit neural tissue regeneration after SCI. If RACs are successfully induced to differentiate into neurons artificially in lesion site of spinal cord, it will be beneficial for restoring spinal cord functions. At previous studies in vitro, we observed that the expression level of miR-124 was significantly lower in RACs compared with neurons, and RACs expressed high-level Sox9 and NFIA predicted to be the target genes of miR-124, also Hes1 gene of a Notch1 signaling downstream major target. We suppose that Notch1-Hes1 pathway is up regulated in RACs, and if inhibiting Sox9 and NFIA genes translation at post transcription level by over expressing exogenous miR-124, it can effectively down regulate the expression of Hes1, and promote neural differentiation of RACs, which will attenuate the glial scar formation in vivo. In this study, we will isolate and purify RACs from lesion sites of rat’s spinal cords and investigate the efficiency of RACs trans-differentiation into neurons after artificially over expressing miR-124 and inducing to neuron in vitro. Then, to induce the over-expression of miR-124 in RACs at the spinal cord lesion site, we will develop a lentivirus-mediated miRNA delivery system using the promoter of glial fibrillary acidic protein (GFAP), and observe the neural differentiation and the glial scar formation of RACs which is specifically over expressed miR-124 after lentivirus infection. Finally, we will reveal the mechanisms that the way miR-124 regulates Hes1 expression. This study will provide new experimental clues and data about how to promote neural differentiation potential of RACs and enhance SCI recovery through targeting RACs in vivo in future.

反应性星形胶质细胞（RACs）具有神经干细胞的部分特征，但在体内并未向神经元分化，其形成的胶质瘢痕却成为脊髓损伤治疗的瓶颈，因此诱导RACs分化为神经元对脊髓损伤的修复有重要意义。申请人前期研究显示，RACs低表达miR-124，但高表达Sox9、NFIA和Notch1通路的主要信号分子Hes1。因此假设外源性miR-124通过调控Sox9和NFIA，抑制Notch1通路，有可能促进体内RACs向神经元分化并减少胶质瘢痕的形成。本项目拟首先在体外研究过表达miR-124对从脊髓损伤部位获取的RACs向神经元分化的影响；进一步应用脊髓损伤模型，在体内原位确认靶向高表达miR-124 的RACs向神经元分化和胶质瘢痕形成减少的形态学依据。最后，通过探索miR-124的作用机制，揭示RACs内调控Hes1表达的分子调控模式，为脊髓损伤再生修复的研究提供新思路和实验数据。

中枢神经系统损伤时星形胶质细胞（ACs）活化为反应性星形胶质细胞（RACs）与小胶质细胞、巨噬细胞等形成致密的胶质瘢痕，脊髓组织再生修复。因此，如何抑制和消除RASs形成的胶质瘢痕成为治疗脊髓损伤的科学难题。. 从中枢神经损伤部位分离培养的RASs在体外具有神经干细胞特点。可是这些细胞移植到体内后并未分化为神经元反而只分化为ASs和少突胶质细胞。如果人为地使RACs在体内诱导分化为神经元，这不仅减少胶质瘢痕的形成，还利于神经网络的再形成和神经组织功能的恢复。基于RACS显著低表达神经元分化中发挥关键作用的microRNA-124 (miR-124)，在本项目中我们人为提高了RACs中的miR-124含量，结合Ruxolitinib、SB203580 和Forskolin等小分子化合物组合，应用大鼠乳鼠大脑皮层星形胶质细胞研究了RACs向神经元诱导分化的可行性，分析了诱导分化获得的神经元的表型、种类以及与正常乳鼠大脑神经元比较高通量测序分析了mRNA表达谱，并分析了诱导RACs为神经元的过程中miR-124的作用机制。同时，还进行了其他两项研究：①研究了人为高表达miR-124的雪旺细胞（SCs）与ACs的相容整合和高表达miR-124的SCs对RACs胶质瘢痕形成的影响；②研究了miR-124对脊髓损伤部位分离培养的成纤维细胞I型胶原表达以及成纤维细胞增殖的影响。. 研究结果显示：①miR-124结合Ruxolitinib等小分子化合物组合显著诱导RACs分化为神经元；②这一组合还有效诱导乳鼠脊髓RACs分化为神经元；③高通量mRNA测序分析显示，诱导获得的神经元与正常乳鼠大脑皮层神经元有相似的表达谱；④miR-124通过抑制Sox9和NFIA的蛋白表达下调Hes1蛋白的表达参与调控RACs为神经元；⑤高表达miR-124的SCs促进了与ACs的相容整合也部分抑制了RACs瘢痕形成的特性；⑥miR-124显著抑制了脊髓损伤部位分离的成纤维细胞I型胶原蛋白的表达以及细胞增殖。. 以上研究结果为以miR-124为关键分子诱导RACs为神经元减少胶质瘢痕形成，减少纤维瘢痕形成促进脊髓组织再生修复以及以miR-124改造SCs提高脊髓损伤的治疗效果，进行了有意义的探索，为以miR-124治疗脊髓损伤治疗的研究提供了理论依据和实验数据。