神经再生和神经环路重塑障碍是脊髓损伤瘫痪的主要原因，损伤后神经元存活、再生及环路重建与局部微环境密切相关。VEGF能促进细胞分裂，参与新生血管形成和细胞增殖、生长、存活等多种过程，但其在神经再生、保护中的作用机制尚不明确。我们预实验发现：新生鼠脊髓全横断后2月，后肢运动功能恢复、流式细胞仪筛查出VEGF显著性升高、MAPK磷酸化明显增加。鉴于VEGF可介导MAPK通路调节神经细胞的生长、分化、存活，本项目提出：VEGF介导MAPK信号通路可以改善脊髓全横断新生鼠的局部微环境，促进脊髓内部神经环路重塑并恢复运动功能。我们拟对脊髓全横断新生鼠进行VEGF干预，研究脊髓局部微环境变化、脊髓神经环路可塑性和功能恢复。研究结果将从形态结构、功能评价和细胞分子信号三方面阐述VEGF的作用机理，成果为脊髓损伤患者的修复治疗提供理论依据，也为探索新的靶点治疗奠定基础。

The obstacle of neural regeneration and circuit reorganization is the leading cause of motor paralysis in spinal cord injury. It is found that neuronal survival and neural loop reconstruction are highly correlated with the local microenvironment after injury. Vascular Endothelial Growth Factor (VEGF) is mitogen-activated protein, participating in new blood vessel formation and the cellular processes of proliferation, growth and survival, but its role in nerve regeneration and neural protection is not clear. In our previous study, we found that hind limbs motor function has been restored in neonatal rats after completely transected spinal cord injury. Furthermore, we demonstrated that the expression of VEGF was significantly increased and the phosphorylation of MAPK had higher too, which was correlated with VEGF in microenvironment. VEGF can regulate neuron growth, differentiation, and survival by mediating MAPK signaling. Thus, our hypothesis is: VEGF may affect spinal microenvironment, which enhances plastic changes of propriospinal circuit and restores behavioral function in neonatal rats after completely transected spinal cord injury through mediating MAPK signaling pathways. To test this hypothesis, we will study microenvironment changes, the plastic changes of propriospinal networks and functional recoveries in neonatal rats after completely transected spinal cord injury by VEGF intervention, which will illustrate the mechanisms of VEGF from the morphology, function evaluation and related molecular signaling. If successful, this research will provide theoretical basis for the repair treatment of patients with spinal cord injury, and also lay a foundation for exploring new targets for therapy.

VEGF介导MAPK信号参与新生鼠脊髓T9全横断后自发性运动功能的恢复，本课题通过阻断VEGF受体的活性和给予重组蛋白过表达VEGF的体内和体外实验，观察动物行为功能学、运动神经元、脊髓腰骶节段的神经环路以及细胞信号分子的变化，对VEGF参与新生鼠脊髓全横断后自发性运动功能恢复的机制进行了初步探讨。首先通过bio-plex试剂盒筛选处出新生鼠（P1）与成年鼠（P28）脊髓全横断后脊髓内差异的细胞因子，明确新生鼠损伤后VEGF较成年组显著性高表达。进而在新生鼠中应用VEGF受体阻断剂阻断VEGF受体的活性和在成年鼠中腹腔注射重组蛋白VEGF，发现VEGF治疗T9脊髓全横断P28可显著改善其运动功能恢复；而VEGF受体抑制剂处理T9脊髓全横断P1，可显著减缓其自发性功能恢复。PRV逆行标记和免疫荧光显示，P28损伤后，脊髓腰段内神经元存活数目显著减少和局部神经环路重塑较差，比P1组更严重。VEGF治疗P28可增加神经元存活数量，改善其神经环路重构。VEGF抑制剂可致P1损伤后L3节段神经元死亡增加，神经环路重构减少。体外培养的大鼠胚胎脊髓运动神经元及脂多糖(LPS)诱导实验发现，VEGF干预可减少的神经元凋亡，促进了神经突起的生长。Western blots分析脊髓腰段和培养的脊髓运动神经Erk1/2磷酸化水平的，LPS诱导炎症损伤后的Erk1/2磷酸化水平较对照组低。VEGF治疗上调Erk1/2磷酸化水平，VEGF抑制剂下调Erk1/2磷酸化水平。综合上述结果，本研究为VEGF通过Erk信号调节脊髓全横断后炎症反应、促进神经元存活和神经环路重建、改善后肢运动功能恢复，为脊髓损伤患者的修复治疗提供理论依据，也为探索新的靶点治疗奠定基础。项目资助发表了SCI论文1篇。培养硕士生3名，其中1名已经取得硕士学位，2名在读。项目投入经费10万元，支出10.0万元，各项支出基本与预算相符。