Reelin/GSK-3β/NR2B信号通路在调节神经性疼痛中的作用研究

The upregulation of NMDA receptor NR2B subunit is one of the main mechanisms contributing to neuropathic pain. However, the underlying mechanisms for the upregulation of NR2B subunit are still poorly understood. Reelin is mainly secreted by GABAergic neurons in the central nerves system. Recent studies shown that Reelin participated in the inhibition of GSK-3β and downregulation the expression of NR2B subunit. Previous studies revealed that peripheral nerve injury caused significant GABAergic neurons apoptosis. These studies indicated that GABAergic neurons apoptosis may cause insufficient Reelin production and further upregulate the expression of NR2B subunit. In this study, Reelin expression vector and RNA interference will be used to modulate the expression of Reelin in the spinal cord of rats. Pharmacologic interference, RT-PCR, Western blot and immunohistochemisty will be used to explore the role of Reelin/GSK-3β/NR2B signal pathway in the modulation of neuropathic pain. This study will elucidate the contribution of Reelin/GSK-3β/NR2B signal pathway in neuropathic pain and confirm the apoptosis of GABAergic neurons resulting in the insufficient production of Reelin which further upregulate the expression of NR2B subunit in the spinal dorsal horn. These studies will provide theoretical support for treatments of neuropathic pain targeting Reelin.

脊髓水平NMDA受体NR2B亚单位表达上调是神经性疼痛的主要机制之一，但引发因素仍不清楚。在中枢神经系统，Reelin主要由γ-氨基丁酸能（GABAergic）神经元产生，最近研究显示其参与抑制GSK-3β并下调NR2B的表达。外周神经受损后，GABAergic神经元发生明显凋亡。这提示GABAergic神经元凋亡导致Reelin产生不足可能参与NR2B的表达上调。本研究采用Reelin表达载体和RNA干扰的方法调节Reelin在脊髓水平的表达，利用药物干预、RT-PCR、Western blot及免疫组化技术对Reelin/GSK-3β/NR2B信号通路进行研究。本研究将阐明脊髓背角中GABAergic神经元凋亡与NR2B表达上调的关系，确定Reelin/GSK-3β/NR2B 信号通路在神经性疼痛中的作用，为临床以Reelin 为靶点治疗神经性疼痛提供理论依据。

Reelin 是一种细胞外基质大分子糖蛋白，在调节神经发育和突触的功能中起着重要的作用。以往的研究发现，reelin缺失小鼠表现疼痛行为异常和传递疼痛和伤害性刺激的脊髓中枢出现紊乱。在神经元迁移结束后，脊髓尤其在脊髓背角的浅层有较高的reelin表达。这提示，reelin可能在传递疼痛和伤害性刺激过程中起着其他的作用。但是前期的研究都受到了reelin参与神经元迁移而影响神经发育的影响。为了探讨成年大鼠脊髓背角中reelin缺失对神经构造正常大鼠的影响，我们采用了鞘内注射介导reelin shRNA的慢病毒载体（lentiviral vector-mediated shRNA against rat reelin, LV-shRNA-Reelin）的方法来沉默reelin的表达。我们的研究发现，鞘内注射LV-shRNA-Reelin对假手术组大鼠的双侧后肢足底和坐骨神经结扎诱导的慢性神经性疼痛大鼠的对侧后肢足底的疼痛行为都没有明显的影响。但是，我们的研究发现，坐骨神经结扎大鼠的同侧脊髓背角中reelin的表达明显降低。同侧脊髓背角中GSK-3β的活性和NR2B的表达水平都明显上升。Reelin基因沉默可进一步加重同侧后肢足底的热痛觉过敏和机械性痛觉超敏。同侧脊髓背角中GSK-3β的活性和NR2B的表达水平也进一步上升。预先注射GSK-3β的抑制剂LiCl或TDZD-8都明显逆转由鞘内注射LV-shRNA-Reelin引起的神经性疼痛加剧和NR2B表达上调。总之，这些结果提示：生理状态下，reelin对传递疼痛和伤害性刺激过程无明显作用。Reelin表达减少可能失去GSK-3β的抑制而上调脊髓NR2B的表达进而参与外周神经损伤（坐骨神经结扎）引起的神经性疼痛过程。