Slit-Robo信号转导通路对周围神经损伤后修复机制的研究

Peripheral nerve injuries are common in both civil and military environments and are primarily the result of transection injuries or burns. These injuries typically lead to the development of neuropathic pain and life-long loss of motor and sensory function in patients. It is a large-scale problem annually affecting more than one million people worldwide with a significant socioeconomic impact and greatly compromises the quality of life of affected individuals. Following an injury, nerve stumps on either side retract and generate a gap which must be bridged by microsurgeries. Autograft is the current gold standard for bridging the gap. However,less than 50% patients have evidence of full functional recovery after autograft and the reasons for poor functional recovery are largely unknown. Recently, we have successfully applied the whole mount staining on transected mouse sciatic nerve and we have observed significant populations of axons growing outside of both the proximal and distal stumps. Our new technique has revealed that axonal guidance defects around the bridge contribute significantly to the poor functional recovery of autografts in patients. More importantly,our current work from Schwann cell specific Sox2 knockout mice, has found a remarkable phenotype with a significant population of axons leaving the bridge after sciatic nerve transaction. Further study by gene microarray, RT-PCR and Western blotting has confirmed that Sox2 up-regulates the Robo1 receptor in Schwann cells after sciatic nerve injury which led us to investigate the role of Slit-Robo signaling pathway in peripheral nerve regeneration. Out preliminary data has revealed that Slit1-3,Robo1-2 and Robo4 are dynamically expressed in the adult peripheral nervous system before and after injury. Particularly,Slit2 and Slit3 are secreted by cells surrounding the bridge to interact with Robo1 in Schwann cell leading processes and also with Robo2 in the growth cones of sensory neurons to keep regenerating axons in the bridge. Our strong data together with our expertise in PNS regeneration and the whole mount staining technique put us in a unique position to address the roles of Slits and Robos in peripheral nerve regeneration. We have obtained the permissions from Dr William Andrews (University of London), Prof Marc Tessier-Lavigne (Rockefeller University) and Prof David Ornitz (Washington University) for using their Robo1-2, Slit1-3 global and conditional knockout mice to investigate their functions in axonal re-growth; motor and sensory function recovery; neuronal survival and angiogenesis; Schwann cell migration,de-differentiation,proliferation and remyelination. We will also construct nerve guidance conduits loaded with recombinant Slit1-3 and Robo-FC and study their effects on peripheral nerve repair in vivo. We hope this novel approach will provide the opportunity to develop new therapeutic strategies for improving peripheral nerve repair following trauma.

周围神经损伤在各类事故中频繁发生，严重损伤如神经断裂必需进行临床手术，但50%以上的病人手术后功能恢复较差。本实验室最新的研究结果表明，神经轴突导向因子Slit对成年周围神经修复起十分关键的调节作用。该项目将以坐骨神经损伤为模型来进一步完善Slit及其受体Robo在周围神经系统损伤前后的表达模式及动态变化，并从英、美两国引进Robo1-2和Slit1-3基因敲除小鼠深入研究Slit-Robo信号转导通路对再生轴突和雪旺氏细胞生长锥的生长及雪旺氏细胞迁移的导向作用，对轴突再生速率、运动和感觉功能的恢复、神经元的存活及雪旺氏细胞去分化和增生的影响，对雪旺氏细胞与轴突相互作用及神经血管再生的调节作用。全面阐明Slit-Robo信号转导通路对周围神经修复的作用机制对开展其它神经轴突导向因子的研究具有理论和实验指导作用，更有望将Slit1-3和Robo-FC用于临床促进病人运动和感觉神经功能的恢复。

周围神经损伤在各类事故中频繁发生，严重损伤如大间隙神经断裂必需进行临床手术修复，自体神经移植是目前对大神经间隙修复的最好方法。由于移植神经源的严重缺乏，国内国际研究者们大力开发了可降解人工合成神经导管并结合干细胞的使用来替代自体神经移植，但临床上使用人工合成神经导管和干细胞的结果表明，这些修复方法对病人功能恢复的促进作用与预期效果仍然存在着很大的差距。神经导向缺陷是目前临床上使用这些修复方法导致病人功能恢复较差的主要原因。在胚胎发育的过程中，已发现多种神经轴突导向分子对整个神经网络的形成起十分重要的调控作用。本课题组和其它研究小组都报道了这些神经轴突导向分子及其相应受体在成年周围神经系统中持续表达或因神经损伤而被诱导表达。因而致力研究这些神经轴突导向分子对周围神经再生的促进作用有望开发新的治疗途径来促进病人功能的恢复。本项目以Slit家族神经轴突导向分子为研究起点，采用原位杂交，定量PCR和蛋白质印迹等室验方法，首先研究了Slit1，Slit2和Slit3及其受体Robo1和Robo2在成年小鼠周围神经系统中的表达模式。然后以坐骨神经切断损伤为实验模型研究了Slit1-3和Robo1-2在成年小鼠周围神经损伤后表达的动态变化。我们发现在围神经损伤后，Slit1和Slit2 的表达显著下调而Slit3的表达在神经桥中却显著上调。免疫荧光标记表明Slit3在神经桥外周的大量巨噬细胞中高表达，而Robo1 和Robo2在再生神经轴突中表达。Robo1同时在神经桥内的迁移性雪旺氏细胞中高表达。本项目进一步以坐骨神经切断损伤为实验模型，在Slit1-3和Robo1-2基因敲除小鼠中研究了Slit1-3和Robo1-2对周围神经再生的调节作用。研究结果表明，Slit3和Robo1基因敲除小鼠存在明显的再生轴突定向生长和雪旺氏细胞定向迁移的缺陷，这表明巨噬细胞起源的Slit3在神经间隙中主要对再生神经轴突的定向生长和雪旺氏细胞的定向迁移起通道性排斥导向作用。本项目的开展不但明确了Slit-Robo信号转导通路对周围神经损伤后修复的作用机制，而且为我们进一步研究其它神经轴突导向分子对周围神经损伤后的修复作用打下了坚实的实验与理论基础，更为今后开发含混合型神经轴突导向分子的新型神经导管用于临床来促进病人功能恢复而铺开道路。