Tmem100调控TRPV1-TLR4神经免疫复合体在慢性盘源性疼痛中的作用机制研究

Low back pain caused by disc degeneration was one of the most common problems in Department of Orthopaedics.73% of people had severe discogenic low back pain in his or her life with uncertain therapeutic efficacy and delayed recovery. In the pre-experiment, we had constructed an expression vector with functional TAD peptide, and then it was transfected to dorsal root ganglion(DRG) of the rats with the function of interference of C terminus of TRPV1.Tmeme100, which was related to the development of intestinal nervous system, was found to be expressed in DRG neuron, and was co-localized with TRPV1 and TLR4.Therefore, we proposed a hypothesis that Tmem100 as an important pain-regulating protein, formed a neuroimmune complex with TRPV1 and TLR4, and contributed to chronic discogenic pain. We aimed to start the experiment around cell and animal models, using many methods and techniques such as immunoprecipitation, electrophysiology, etc. We will clarify the specific binding sites of Tmem100 with TRPV1 and TLR4, and the expression and function of Tmem100 and TRPV1-TLR4 neuroimmune complex in chronic discogenic pain, and then demonstrate the mechanism of competitive inhibition of the neuroimmune complex for pain relief and regulation of the immune response after TAD transfection. We firmly believed that it will be valuable to provide a new way for treatment of chronic discogenic pain in the future.

椎间盘退变引起的腰背痛是骨科最常见的疼痛之一，73%的人一生中会出现严重的盘源性腰背痛，治疗效果不确切，极易迁延不愈。我们前期转染TAD多肽表达载体至大鼠背根神经节（DRG），可特异性干扰TRPV1 C端结构域蛋白结合位点功能，同时发现与肠道神经系统发育有关的Tmem100在DRG神经元中有表达，且与TRPV1及TLR4均存在共同表达。因此提出假设Tmem100作为一种重要的疼痛调节蛋白，与TRPV1及TLR4形成神经免疫复合体，在慢性盘源性疼痛中起着重要的作用。从细胞及动物模型两方面入手，借助免疫共沉淀、电生理等技术展开实验，明确Tmem100与TRPV1、TLR4特异性结合位点，阐明Tmem100与TRPV1-TLR4形成的神经免疫复合体在慢性盘源性疼痛中的表达及作用，并通过TAD多肽竞争抑制神经免疫复合体形成，探明其缓解慢性盘源性疼痛并调控免疫应答的机制，为盘源性疼痛治疗提供新思路。

随着人口老龄化，脊柱退行性疾患发病率高达28%，仅腰椎间盘突出症，我国每年新发病例近 2000万，其诊断诊疗是脊柱外科医生面临的巨大挑战。目前，脊柱退变病因机制复杂，细胞凋亡及神经病理性疼痛等机制尚未阐述清楚。因此，寻找椎间盘退变及其慢性疼痛调控的关键机制及早期有效生物学治疗具有重要意义。本项目在慢性疼痛机制、载体构建、病毒转染、动物实验等方面取得一定成果。此外，进行了椎间盘退变相关基础研究以及纳米材料在骨科中的应用探索，最大发挥本课题的作用。.本课题紧紧围绕椎间盘退行性疾患及其相关神经病理性疼痛为研究重点，前期转染TAD多肽表达载体至大鼠背根神经节（DRG），特异性干扰TRPV1 C端结构域蛋白结合位点，从而缓解神经病理性疼痛。从细胞及动物模型两方面入手，明确Tmem100在背根神经节神经元及脊髓背角中表达情况，发现其在神经损伤所致疼痛中表达降低，比较不同疼痛模型下（CCI、TNI）Tmem100差异性表达情况，同时构建AAV6-GFP-Tmem100表达载体，采用创新的微创注射转染技术，观察过表达Tmem100可缓解慢性神经病理性疼痛；同时，阐明了构建的新型多肽CBD3通过有效阻断N型电压门控钙通道（CaV2.2）活性缓解神经病理性疼痛中的重要作用及机制；针对引致慢性腰背痛的椎间盘退行性疾患，结合纳米纺丝新型材料，3D打印并构建含有尿源干细胞（USCs）外泌体的生物活性支架，验证尿源干细胞USCs外泌体可有效抑制压力应激引起的髓核细胞凋亡，揭示了内质网应激在椎间盘退变中的重要作用。同时，进一步研究发现富含MATN3的USCs外泌体在体内、外均有效促进细胞外基质合成从而缓解椎间盘退变，该项目研究成果有利于发现治疗椎间盘退变及其慢性神经病理性疼痛的新靶点和重要调控机制，为临床使用USCs或外泌体延缓甚至逆转椎间盘退变奠定坚实前期研究基础，也为神经病理性疼痛基因治疗临床应用奠定基础。

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