SCN9A在外周神经结扎诱发的镜像痛中的机制及与胶质细胞信号传导的双相作用

To date, the mirror-image hyperalgesia mechanism is unclear yet. It may be related to humoral immunity, peripheral nerve injury, central sensitization, cortical downstream regulation. Nowadays the research interests are focusing on the involved signal pathway of glial cells during the process of mirror-image pain.SCN9A encoding a subunit of voltage-gated channel Nav1.7, in which a single-gene mutation is closely related to congenital abnormal pain. In our preliminary studies we find SCN9A abnormality highly expressed in ganglion with the development of mirror pain. In the current study, we hypothesized that peripheral nerve damage might trigger high expression of SCN9A in ipsilateral spinal ganglia, and lead to the activation of spinal cord glial cell. After that, the expression of SCN9A might be higher in contralateral spinal ganglia which will result in the mirror pain. To prove the hypothesis, a rat mirror-image pain model established by nerve distal ligation is used in this study. RT-PCR, Western-blot and immunohistochemistry were used to test the SCN9A expression in spinal ganglion and the activation of signal transduction pathway of glial cells in spinal cord as well as threshold changes of detected pain in rat model at different time points. Then after transected spinal cord , respectively high inhibited the activation of glial cell and expression of SCN9A gene to observe the biphasic effect between central immune regulation by glial cells and expression of SCN9A to verify the hypothesis .

目前为止，镜像痛敏发生的机制尚不清楚，可能与体液免疫、外周神经损伤、中枢敏化、皮质下行调节、以及目前国际上关注的热点-胶质细胞的参与的信号传导相关。本项目关注的基因SCN9A编码电压门控Nav1.7通道的ａ亚基，其单基因突变与先天性疼痛异常密切相关。在前期研究SCN9A与神经病理痛的相关机制时，发现神经结扎模型的镜像侧脊神经节SCN9A异常高表达，且其高表达与镜像痛的发生发展有密切关系。因此本项目提出假说：周围神经损伤- - 同侧脊神经节SCN9A高表达- - 脊髓胶质细胞活化-对侧SCN9A高表达- - 镜像痛。本项目拟神经远端结扎制作大鼠镜像痛模型，通过RT-PCR、Western-blot、免疫组织化学等方法检测脊神经节不同时间点SCN9A表达及脊髓胶质细胞的信号传导通路的激活，同时检测模型鼠痛阈的变化；继而分别抑制胶质细胞活性及SCN9A基因的表达，观察二者变化的相互关系，以验证该假说。

本研究采用大鼠神经损伤模型，利用行为学检测、大鼠蛛网膜下腔给药、qPCR、Westernblot、免疫组织化学、细胞培养等方法 ，我们发现：（1）大鼠L5脊神经结扎SNL模型的确可以引起同侧神经病理性疼痛和对侧镜像痛。（2）qPCR和western 显示大鼠SNL模型可以引起双侧Nav1.7表达增高，免疫组织化学、qPCR和western显示SNL模型大鼠脊髓CX43表达增加，星形胶质细胞的活化及双侧神经节卫星胶质细胞活化。（3）大鼠鞘内注射星形胶质细胞抑制剂氟代柠檬酸或CX43抑制剂甘珀酸均可抑制SNL大鼠同侧及对侧痛。（4）SNL大鼠损伤侧DGR显微注射Si-SCN9A不仅可以缓解同侧神经病理性疼痛，而且逆转脊髓的中枢敏化（抑制星形胶质细胞的活化）,并减少对侧神经节内SCN9A表达Nav1.7。上述结果证实了我们提出“外周神经的损伤---- DRG内Nav1.7表达升高-----脊髓中枢敏化-----对侧DRG内Nav1.7表达致镜像痛”的假说，为神经病理性损伤导致的疼痛的机制和治疗提供新的思路。