S100β基因修饰的嗅鞘细胞修复周围神经缺损的机制

Peripheral nerve injury (PNI) is common and resulted in considerable long-term disability，but the clinical outcome is often not optimal. Although some positive effect was get by combining the bridged material of no antigen with olfactory ensheathing cells (OECs) transplantation to treatment the PNI, the method was limited by the transplanted OECs' survival number under the inflammation condition, which would lead to the poor repairing effect. S100β plays the important role in cell proliferation and maintaining the axonal structure of neuron, it may also decrease some proinflammation factor, but its detail molecular function is still unclear. We have found the low expression level of S100β in the transplanted part, when utilized the bridged material of no antigen with olfactory ensheathing cells to repair the sciatic nerve injury at the acute inflammation phase. In our study, we want to check whether the S100β gene modified OECs combining with the bridged material of no antigen can promote the survival of donor cell (OECs) and the function of the host neuron, and then we will clarify the underlie mechanism. The molecular role of S100β in the cellular survival ability and the spinal ganglia cell axonal extendedness will be checked with an established in vitro inflammation model. The viability of OECs will be measured by MTT; the apoptosis will be evaluated by TUNEL staining and gene expression of apoptosis related factor; the expression of proinflammation related factor and S100β will be checked by real time PCR and ELISA; the axonal proliferation related protein will be check by immunocytostaining. Then the involvement of S100β in the NFκB/MAPK signaling pathway will be analyzed. The transcriptional factors will be check by western blot. The repairing effect of the S100β gene modified OECs combining with the bridged material of no antigen will be evaluated after grafting them to the rat sciatic nerve transection model by measuring the recovery of function, and then the underlies mechanism will be analyzed. What our finding in present study will be provide the possible method and fundamental theory for clinical effective treatment to PNI.

嗅鞘细胞（OECs）移植对治疗周围神经损伤具有积极的效果，但其移植后在炎症条件下的低存活率限制了其神经修复功能的发挥。在周围神经缺损的急性炎症期，低水平S100β的微环境可能影响移植后OECs的生存及其促进缺损远端神经轴突功能恢复的能力。本项目拟利用S100β转染的OECs培养体系（S100β(+)OECs）和S100β(+)OECs/神经节细胞共培养体系，通过体外炎症模型，研究S100β(+)OECs在炎症环境下的生存能力及其对周围神经元的影响，探讨S100β通过NFκB/MAPK及RAGE信号通路调控神经细胞增殖的机制；将S100β(+)OECs联合异种神经桥接体移植于大鼠坐骨神经缺损模型，对比观察S100β(+)OECs存活状态和生物活性，探讨其对周围神经损伤急性炎症期的修复效果及其机制。阐明S100β对神经细胞生存微环境的影响及机制，将为临床治疗周围神经损伤提供可能的治疗手段。

外伤、手术等原因引起的周围神经损伤在临床上十分常见。目前各种周围神经修复方法仍然存在一定的局限性，使神经修复的效果不理想，特别是神经损伤后的功能恢复至今尚未达到令人满意的结果，严重地影响了患者的生活质量。目前，有效的修复周围神经缺损仍是亟待解决的难题。近年来，国内外学者对周围神经损伤的修复技术做了大量实验及临床研究，这些研究均围绕着种子细胞的采集与扩增、桥接体材料的选择以及神经营养因子的影响等方面展开。运用分子生物学方法，从分子水平调控桥接体移植后的微环境及细胞行为的研究一直是学术界关注的方面。.嗅鞘细胞（OECs）移植对治疗周围神经损伤具有积极的效果，但其移植后在炎症条件下的低存活率限制了其神经修复功能的发挥。在周围神经缺损的急性炎症期，低水平S100β的微环境可能影响移植后 OECs 的生存及其促进缺损远端神经轴突功能恢复的能力。 本项目建立并利用 S100β转染的 OECs 培养体系（S100β(+)OECs）和 S100β(+)OECs/神经节细胞共培养体系，通过体外炎症因子IFN-γ诱导的模型，证实S100β可以下调IFN-γ激活的炎症模型中炎症因子的基因表达水平，减少促凋亡因子的表达，有利于嗅鞘细胞存活发挥作用。进一步证实S100β蛋白可能通过介导 NFκBp65/MAPK p38 通路调控炎症环境中 OECs 的炎症反应机制。在急性炎症条件下，OECs可以促进DRG细胞突起的生长，而S100β-OECs对DRG细胞突出的生长有更为明显的促进作用。S100β-OECs可能通过CX3CR1-JNK通路参与对DRG的功能调控。建立动物移植模型，通过检测坐骨神经运动，感觉等功能的恢复情况，证实 S100β 基因修饰的嗅鞘细胞联合去抗原性异种神经桥接体移植对坐骨神经缺损急性炎症期的修复有明显效果，为临床的急性炎症期周围神经缺损修复治疗寻找可能的方法及理论依据。