新型镇痛靶点α2δ-1-NMDAR稳定表达细胞株构建及镇痛药物筛选的应用

A novel cell tool for α2δ-1-NMDAR target-based analgesic drug discovery

摘要:

在多种神经性疼痛动物模型中均发现周围神经损伤可导致由CACNA2D1编码的α2δ-1蛋白在脊髓和背根神经节(DRG)中表达显著上调。CACNA2D1过表达使脊髓背角神经元的突触前和突触后NMDAR活性增强，引起疼痛过敏症。α2δ-1与NMDAR相互作用，促进了NMDAR在细胞表面转运和突触膜上的定位，可引起神经病理性疼痛，且α2δ-1-NMDAR复合物也被发现存在于其他多种病理状态下，如某些神经源性的高血压、脑缺血。本研究使用慢病毒转染构建稳定表达α2δ-1-NMDAR复合物的人肾胚HEK293T细胞株；通过荧光显微镜、RT-qPCR及Western blotting方法对所建立的稳转细胞系进行鉴定，结果显示HEK293T被成功转染且基因能够稳定表达；并采用膜片钳技术成功建立该细胞株靶向药物筛选体系。该细胞株为进一步研究α2δ-1-NMDAR复合物的相互作用机制及针对慢性疼痛和相关疾病的低副作用的药物筛选提供了良好应用前景的细胞模型。

Abstract:

The α2δ-1 protein coded by Cacna2d1 is dramatically up-regulated in dorsal root ganglion (DRG) neurons and spinal dorsal horn following sensory nerve injury in various animal models of neuropathic pain.Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity.The α2δ-1-NMDAR interaction promotes surface trafficking and synaptic targeting of NMDARs in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury,as well as in other pathological conditions such as in the paraventricular nucleus (PVN) with neurogenic hypertension and in the brain with ischemic stroke.The lentiviral transfection method was used to construct a human embryonic kidney HEK293T cell line that could stably express α2δ-1-NMDAR complex.A stably transfected cell line was observed by florescence microscope,and identified by RT-qPCR and Western blotting.The results showed that the HEK293T cell line was successfully transfected and the genes could be stably expressed.Subsequently,the transfected cell line was successfully developed into a target drug screening system using patch clamp techniques.It provides a promising cell model for further research on the interaction mechanism of α2δ-1-NMDAR complex and drug screening for chronic pain and related diseases with low side effects.