基于背根节-脊髓背角神经元图形化网络的慢性痛机制研究

Chronic pain is recognized as an important problem in clinic, but its pathogenesis is not well understood. In previous work, we have found that mechanically-gated TRPN/4 channel mediate noxious stimli-evoked currents in nociceptive neurons based on Caenorhabditis elegans model. In this proposal, we will study the role of mechanically-gated ion channels in chronic pain based on primary neuronal cultures of rat. Synapses between dorsal root ganglion neurons and spinal cord dorsal horn neurons are the first loci for transmission of pain. However, there is a lack of reliable model that reconstitutes synapse between these two populations. To co-culture dorsal root ganglia neurons and dorsal horn neurons of rats in vitro and mimic in vivo morphology, microsystem technology will be used to modify the surface of substrates, and special pattern will be designed to guide the location of soma and the growth direction of axon. Based on this patterned neuronal network, we will utilise molecular-biology, electrophysiology, and calcium imaging technologies, to characterize potential ion channels and factors mediating chronic pain and to investigate how neurons communicate. Our studies lay the foundation for the future work which elucidates the molecular mechanisms underlying chronic pain in mammal, and may advance our understanding of chronic pain from a new perspective, and reveal new targets for treatment.

慢性痛是普遍存在的重要临床症状，目前发病机制尚不清楚。我们前期基于秀丽线虫的工作鉴定了机械门控的TRPN/4通道在伤害性感受（痛觉）中的作用，本研究将进一步在哺乳动物细胞水平上进行机械门控离子通道在慢性痛中作用的研究。背根节神经元和脊髓背角神经元形成的突触连接是痛觉传递的重要部位，但目前缺乏合适的模型对其进行研究。我们将结合微系统技术，对培养神经元的基底进行表面处理，设计特殊的图案控制神经元胞体的位置以及诱导其轴突的生长方向，实现背根节神经元和脊髓背角神经元体外的图形化共培养，用以模拟体内的生理情况。基于此体外模型采用分子生物学、电生理和钙成像等技术，鉴定与慢性相关的离子通道和调节因子，深入研究伤害性感受神经环路中神经元之间的相互通讯。我们的研究将为阐明哺乳动物中慢性痛的分子机制和调控网络奠定基础，从新的角度加深对慢性痛的理解，将为其治疗提供新的靶点。

本研究结合微系统技术，对培养神经元的基底进行表面处理，设计特殊的图案控制神经元胞体的位置以及诱导其轴突的生长方向，实现了皮层神经元体外的图形化培养。结合分子生物学、电生理和钙成像等技术，研究了形态学对神经元功能的影响。图形化培养的皮层神经元只有2个树突，而正常离体培养的皮层神经元平均6个树突。研究结果表明：树突数目的减少影响了神经元的自发放电模式，并且当给予电流刺激时的响应也发生改变。相对正常培养的神经元，图形化培养的神经元自发放电的簇发形式更少，在给予电流刺激时响应的动力学特征也不一样。这一结果提示图形化培养的神经元使其形态特征变简单，并会影响其生理特征，这一变化在今后设计具有计算功能的生物神经元网络需要注意。