三叉神经根区Wnt信号通路参与三叉神经痛发病机制的研究

Wnt signaling pathway regulates the expressions of myelin sheath genes and myelination. Our research team built a novel trigeminal neuralgia (TN) animal model method by chronic compression injury of trigeminal nerve root previously. To evaluate the extent of injury in the TN animal, we are going to use immune electron microscopy technique to observe the myelin ultrastructure; to study the myelin-axon neurotrophins, main Wnt signaling molecules and myelin associated protein expression, using immunohistochemistry, Western blot, in situ hybridization and PCR technologies; and study the abnormal discharges of trigeminal root zone fibers by electrophysiology technique. To further study the regulation mechanism of Wnt signaling pathway to the single culture or co-culture of oligodendrocyte precursor cells and Schwann cells in myelination and remyelination, we are going to use liposomal transfection or lentiviral transfection to overexpress the Tcf4 gene, which is an essential transcription factor of Wnt signaling pathway, and to build the Tcf4 gene knockout cell line by Crispr/Cas9 technology at the same time. Then, engineered nanofibers will be used to instead of axons as myelination stents, the myelin precursor cells regeneration, migration and myelination are studied under the condition of single culture or co-culture of oligodendrocyte precursor cells and Schwann cells. Finally, we are going to assess ethology of TN animal model after intrathecal administration of Wnt inhibitor or Wnt agonist to the TREZ separately, and also the recovery of myelin-axon structure and function. In summary, we want to provide theoretical evidences to explore the pathogenesis of TN, and develop targeted drugs to cure, prevent or even delay the attack of TN.

Wnt信号调控髓鞘相关基因表达和髓鞘形成过程。我们在三叉神经根区（TREZ）慢性压迫诱导三叉神经痛（TN）动物模型基础上，运用免疫电镜观察神经根压迫损伤后髓鞘超微结构改变；免疫组化、Western blot、原位杂交及PCR等技术，研究TREZ神经营养因子和主要Wnt信号分子、髓鞘相关蛋白表达；运用电生理学方法研究神经纤维异常放电。为探讨Wnt信号通路对联合培养的少突胶质前体细胞及施旺细胞的髓鞘形成和再生调控，运用脂质体转染/慢病毒转染过表达Wnt信号通路重要转录因子Tcf4基因，Crispr/Cas9技术构建Tcf4基因敲除细胞系，生物材料纳米丝代替轴突作为髓鞘形成支架，观察髓鞘形成细胞单独培养或共培养条件下再生迁移及髓鞘形成过程。通过TREZ鞘内给药Wnt阻滞剂或激动剂，评估动物行为学和TREZ结构功能恢复情况，为阐明TN发病机制，开发治疗、预防或延缓TN发病的靶向药物提供理论依据。

本项目在三叉神经根区（TREZ）慢性压迫损伤诱导三叉神经痛（TN）大鼠模型上，形态学方法研究TREZ胶质细胞形态和功能改变，发现TREZ中枢神经-周围神经（CNS-PNS）过渡区少突胶质细胞髓鞘和施万细胞髓鞘发生不同程度脱髓鞘改变；星形胶质细胞大量活化增生，从CNS-PNS过渡区中枢端进入周围端，形态学测量显示中枢端星形胶质细胞边界明显变长；周围端施万细胞不同程度活化和再生；髓鞘相关的Fyn蛋白在神经压迫损伤早期降低，后期表达逐步回升；髓鞘碱性蛋白（MBP）表达降低，P75神经营养因子受体（P75NTR）表达增高，神经营养因子BDNF和GDNF在TREZ和TG都发生动态改变。表观遗传学方法研究TREZ区胶质细胞组蛋白乙酰化，发现慢性压迫损伤诱导H3K9、H3K18、H3K27发生不同程度乙酰化并影响大鼠口面部机械痛阈值。TREZ慢性压迫损伤后，CNS-PNS过渡区都有β-catenin表达，主要分布于周围端施万细胞内，β-catenin在术后14 d开始表达上升，28 d逐步降低，星形胶质细胞标记物GFAP与active β-catenin在胶质细胞交界区共表达明显；Wnt 3a、Wnt 5a、Fzd 8等Wnt信号通路分子在神经根压迫损伤后表达上升，其他分子IL-18、TNF-α及C1q表达同步上调。构建靶向敲除β-catenin慢病毒ShRNA局部注射到TREZ后，TN组大鼠口面部机械痛敏下降，IL-18、TNF-α及C1q蛋白表达下调，C3标记的I型星形胶质细胞数量减少，提示靶向敲除β-catenin可抑制星形胶质细胞活化。RNA- seq分析和RT-qPCR结果验证，59个hub基因表达下调，特别是Cd68、Cxcl14、Ccl2、Gpnmb、Hspb1及Lgals3 与TN动物模型密切相关。综上研究，Wnt/β-catenin信号通路参与TREZ慢性压迫损伤后TN周围敏化过程。