血小板反应蛋白4在脊髓损伤后促NG2细胞重编程为神经元中的作用及其分子机制研究

Inhibiting glial scar formation and promoting neural regeneration are key factors for recovery of spinal cord injury (SCI). However, it is difficult for transplanted cell to survive and integrate with injurd tissue after SCI. In contrast, inducing endogenous glial precursor cells in injury sites to transdifferentiate to neurons is more promising. Our previous work demonstrated that EGFR inhibition convertes NG2 cells to neurons and reduces glial scar formation, accompanied with induction of thrombospondin-4 (THBS4). Interestingly, THBS4 overexpression in NG2 cells substantially increased the induction of neuronal markers. The proposal aims to investigate the potential of THBS4-targeting strategy for SCI treatment, by studing the role of THBS4 in promoting NG2 cells to differentiate to neurons and reducing glail scar fromation in SCI. Using gene knockout and overexpression techniques, we planned to elucidate the molecular mechanisms of THBS4-induced neural differentiation of NG2 cells, by analyzing neuron differentiatopm-related signaling pathways, such as Olig2, Neurogenin, ATF6, NOTCH1, FGFR, and AKT. Meanwhile, THBS4-overexpressing lentivirus will be used to infect SCI sites, to evaluate the effect of THBS4-targeting treatment on SCI. Our study will reveal the molecular mechanisms of endogenous glial precursor cells for SCI therapy, and provide reliable evidences for its preclinical application.

抑制胶质瘢痕形成和促进神经再生是脊髓损伤后功能恢复的关键，然而神经元移植到脊髓损伤部位后其存活和与组织整合仍是目前难题。相比，调动内源性前体细胞分化成新的神经元具有巨大优势。我们前期发现抑制EGFR信号可促进NG2细胞转分化为神经元且抑制瘢痕形成，并伴随THBS4（血小板反应蛋白4）的表达上调。该基因过表达后NG2细胞中神经指标显著升高。本课题旨在揭示THBS4基因在脊髓损伤中促进NG2细胞神经分化和减少疤痕形成的作用，探讨其用于治疗脊髓损伤的潜能。我们拟采用基因敲除和过表达等手段，通过对Olig2、Neurogenin、ATF6、NOTCH1、FGFR及AKT等神经分化相关信号通路的分析，研究THBS4促NG2细胞转分化为神经元的分子机理，并利用THBS4慢病毒感染脊髓损伤部位，评价对小鼠脊髓损伤的实际疗效。其结果将揭示内源性前体细胞治疗脊髓损伤的分子机制，为临床前应用提供理论依据。

神经再生是脊髓损伤后功能恢复的关键，调动内源性前体细胞分化成新的神经元具有很大优势。本课题揭示血小板反应蛋白（Thrombospondin-4，THBS4；也称TSP4）基因在NG2细胞分化中的生物学意义。我们对TSP4在NG2细胞重编程为神经元中的作用进行研究，THBS4重组病毒转染NG2细胞（G23）后，NG2细胞中的多个神经元标志物的mRNA和蛋白质表达水平均显著上调，如Tuj1、MAP2、NeuN。相应地，NG2过表达导致星型胶质细胞的标志物GFAP及少突胶质细胞标志物Olig2表达下调。当THBS4基因沉默后，这些标志物的表达趋势刚好相反。对NG2细胞分化过程中转录因子的研究表明，THBS4介导的NG2细胞分化可能与转录因子Ngn1、ATF6、Olig2有关。进一步对TSP4作用的机制进行初步分析，发现TSP4是通过ERK通路调控NG2细胞重编程为神经元。我们的研究结果表明，THBS4可以促进NG2细胞向神经元转分化，为NG2细胞应用于神经损伤的治疗提供有利的理论依据。