谷氨酸能中间神经元桥接皮质脊髓束促进脊髓损伤修复的研究

Voluntary movement dysfunction after spinal cord injury hurdles the rehabilitation effects seriously, and there is still no effective treatments. Reconstructing the injured corticospinal tract through cell transplantation is a promising method for voluntary movement functional repair after spinal cord injury, which has attracted much attention. However, the extent of neural circuits reconstruction after spinal cord injury is highly different when referred to different cell donors, and it is difficult to ensure the therapeutic effects. Based on the literature reports and our previous research, this project aiming at directly differentiating pluripotent stem cells into spinal cord V2a and dI3 neural precursors in vitro and transplanting them combined into the injured rat spinal cord. Afterwards, we explore whether the neural functions after spinal cord injury ameliorate from the neurobehavioral level; or the surviving, migration and differentiation of the grafts from the cellular level; or the extent of the corticospinal tract reconstruction, including the axonal extension and targeting of cell grafts, the corticospinal neurons regeneration and the synapses formation between grafts and hosts from the molecular level through tracer analysis, electrophysiological detection and chemical drug regulation techniques. This will further elucidates the possible role of spinal cord V2a and dI3 glutamatergic interneurons in corticospinal tract reconstruction and neural functions repair, providing a solid theoretical basis for combined cell transplantation in spinal cord injury.

脊髓损伤后的随意运动功能障碍严重影响了患者的康复治疗效果，目前尚缺乏有效的治疗方法。利用细胞移植重建受损的皮质脊髓束环路，是脊髓损伤后随意运动功能修复的很有前景的方法，备受关注。然而，脊髓损伤后移植不同细胞的神经环路重建程度差异性大，难以保证细胞移植治疗效果。结合文献报道及我们的前期研究，本项目拟通过多潜能干细胞体外定向分化为脊髓V2a和dI3神经前体细胞，并联合移植入大鼠损伤脊髓。然后，从动物神经行为学水平观察脊髓损伤大鼠的神经功能有无改善；在细胞水平，探索移植细胞的存活、迁移、分化；并通过示踪分析、电生理检测和化学药物调控技术探讨皮质脊髓束环路重建（包括移植细胞轴突的延伸、靶向连接，皮质脊髓束再生及相互间的突触整合）的情况。以此进一步阐明脊髓V2a和dI3谷氨酸能中间神经元在皮质脊髓束环路重建，及神经功能修复中的可能作用，为联合细胞移植治疗脊髓损伤提供坚实的理论基础。

脊髓损伤后的随意运动功能障碍严重影响了患者的康复治疗效果，目前尚缺乏有效的治疗方法。利用细胞移植重建受损的皮质脊髓束环路，是脊髓损伤后随意运动功能修复的很有前景的方法，备受关注。然而，脊髓损伤后移植不同细胞的神经环路重建程度差异性大，难以保证细胞移植治疗效果。本项目探索了由人胚胎干细胞获得脊髓dI3谷氨酸能中间神经元的化学小分子诱导方法，并提高了其分化效率；利用dI3神经前体细胞移植后，验证其存活，为其移植研究做好充分的铺垫。该研究利用人胚胎干细胞体外定向分化为高纯度的目的细胞平台，实现人多潜能干细胞的饲养层及无饲养层培养，并在特定化学小分子组合的诱导下将其定向分化为高纯度的脊髓神经上皮细胞、dP3神经前体细胞和dI3谷氨酸能中间神经元，完成各阶段细胞的免疫荧光、分子生物学鉴定及神经元的细胞膜片钳检测；同时，通过dI3神经前体细胞的脊髓内定点移植，我们证实了其在体的存活，也进一步验证了其在治疗脊髓损伤的潜力，为后期的细胞移植实验、组织工程等研究奠定了坚实的基础。