Nogo-c基因对周围神经再生及倍数扩增的影响及机制研究

Peripheral nerve injury is a common type of trauma. The prognosis of such trauma is not satisfied, which may impose a heavy social and economic burden on patients. Our team has been concentrating on the basic and clinical research of peripheral nerve injury and regeneration. How to accelerate the growth of the nerve fibers, how to improve the “multiple amplification” effect and improve the target organ’s function after peripheral nerve injury repaired is still a hot research spot in this field. Previous studies showed that the nerve got better functional restoration when using biological conduit small gap sleeve bridging neurorrhaphy.This new method may improve the plasticity, selective regeneration, and multiple amplification of nerve after injury and the target organ got better function recorvery.In the previous experiments, we had observed an interesting phenomenon:if nogo-c gene was knocked-out,the process of peripheral nerve axons regeneration accelerated and the function of target organ recovered better. But, whether peripheral nerve injury can affect the expression of Nogo-C gene? What is the mechanism of Nogo-C gene expression changes in peripheral nerve injury? It is still remained unknown for the influence and mechanism of Nogo-c on the neuron,Schwann cells, regeneration of peripheral nerve, multiple regeneration of nerve when the level of nogo-c expression changes. So this research will mainly study the influence of nogo-c to regeneration of peripheral nerve, then trying to reveal the mechanism of nerve recovery and regeneration, to look for a new passageway to improve the final function after peripheral nerve injury. We hope to solve the long-term clinical problem that the inadequate capacity of peripheral nerve recovery and the delay for peripheral nerve fibers to reinnever target organs.

周围神经损伤是临床上常见的创伤类型之一，其修复效果不佳，带来严重的经济和社会负担。本课题组长期致力于周围神经再生的基础和临床研究。如何加快损伤神经的生长速度和倍数扩增是研究的热点。生物套管小间隙套接法修复周围神经损伤有利于发挥神经的可塑性、再生选择性和倍数扩增等特性，有助于靶器官功能的恢复。前期实验结果显示Nogo-C基因敲除可以加快周围神经轴突的再生速度，促进神经再支配靶器官的功能恢复。但是，周围神经损伤是否会影响Nogo-C基因的表达，影响的机制是什么；Nogo-C基因不同表达水平对神经元、Schwann细胞，周围神经再生、倍数扩增的影响及机制并不清楚。本课题拟通过研究Nogo-C基因对周围神经再生的影响，以期进一步揭示该基因在周围神经损伤修复过程中的作用机制及分子基础并寻求新的促进周围神经损伤修复的切入点。为解决周围神经修复能力不足、神经纤维再支配靶器官不及时等临床难题提供新的思路。

周围神经损伤是临床常见的创伤类型，常造成患者长期远侧肢体功能障碍，严重影响生活质量。本项目利用TALEN技术构建Nogo-C基因敲除小鼠模型，结合同龄野生型小鼠制作坐骨神经钳夹伤模型；Semmes-Weinstein单丝检测盒测试小鼠下肢感觉；足印分析检测小鼠下肢运动功能；腓肠肌湿重测量评估骨骼肌萎缩情况；半薄切片检测坐骨神经纤维数量；透射电镜观察神经髓鞘崩解和再生情况；分离培养原代新生SD大鼠雪旺细胞；细胞水平腺病毒过表达Nogo-C蛋白；Western Blot检测蛋白水平变化；RT-PCR检测mRNA水平变化；Tunel染色检测雪旺细胞的凋亡情况。发现野生型小鼠坐骨神经损伤后，第7天开始到第28天期间损伤神经远端Nogo-C蛋白水平持续升高。Nogo-C基因敲除小鼠坐骨神经损伤后，与损伤的野生型小鼠相比，在损伤后第7天其神经远端细胞凋亡数显著降低、髓鞘清除速度明显增快；坐骨神经损伤后14天，Nogo-C基因敲除小鼠神经纤维再生数量较野生型坐骨神经损伤小鼠明显增多，髓鞘化程度增高，并且靶器官腓肠肌的湿重比显著增加；此外，Nogo-C基因敲除小鼠的运动和感觉功能恢复显著快于野生型小鼠。使用腺病毒在原代雪旺细胞中过表达Nogo-C，可以促进雪旺细胞的凋亡；并且Nogo-C显著降低c-Jun/krox-20表达比，提示抑制雪旺细胞的去分化。表明周围神经损伤后，损伤局部Nogo-C表达升高，升高的Nogo-C促进雪旺细胞凋亡，抑制雪旺细胞去分化能力，从而抑制周围神经再生。