piRNA介导DNA甲基化修饰调控慢性疼痛的新机制

The reason of chronic pain is cellular and molecular biologic changes, result in increasing in neuronal activity and excitability，including alteration of neurotransmitter and receptor in pain signaling pathway. Diversity of clinical methods were used for chronic pain management, but the major reatment in empiric and the curative effect still uncertain, due to the molecular mechanisms underlying chronic pain development remain poorly understood. We found that chronic pain pathological conditions can increase a piRNA located in nucleus what we named piR-004567-L.Functional inhibited of spinal piR-004567-L prevented and reversed thermal hyperalgesia and mechanical allodynia and spinal neuronal sensitization induced by CFA. Overexpression of spinal piR-004567-L in na?ve animals induced pain behavior, but can reversed by 5-AZA or siRNA Dnmt3a. Some evidence has shown that DNA methylation-mediated gene expression modulation contributes to chronic pain, but its functional regulatory mechanisms still remains unknown. We speculate piR-004567-L was involved in epigenetic modification of spinal and regulates chronic inflammation pain. This proposal is based on previous research, we will utilize lentivirus system, RNA-ChIP,, high-throughput sequencing, FISH, qRT-PCR and western blot to elucidate the roles and mechanisms of piR-004567-L regulate DNA methylation in chronic inflammatory pain.

慢性疼痛发生主要原因是脊髓背角细胞和分子学变化，导致神经元活性上调，中枢兴奋性增加，包括传导通路中递质、受体等的变化，而引起痛觉过敏。临床治疗方法多样，但疗效不确切和经验性用药为主，究其原因是对慢性疼痛产生的分子机制不明。申请人发现CFA小鼠炎性慢性疼痛状态下主要在脊髓细胞核表达的mmu-piR-004567-L发生上调表达，鞘内干扰其功能可提高疼痛阈值；过表达导致疼痛阈值下降，但可被DNA甲基化抑制剂5-AZA和小RNA干扰Dnmt3a逆转。已有研究提示DNA甲基化通过调控疼痛相关基因表达介导慢性疼痛产生，推测此piRNA可能通过对DNA甲基化调控参与慢性疼痛过程。本研究在已有研究基础上，采用慢病毒表达、改进RNA-ChIP、高通量测序、RT-PCR、免疫印迹等技术，拟阐明piRNA调控DNA甲基化导致的信号传递及对脊髓背角突触功能的调节机制，为研发理想镇病药物提供新的思路和药物靶标。

目的 伏隔核（nucleus accumbens,NAc）是中脑多巴胺奖赏系统重要的组成部分，因与其他脑区具有广泛的纤维联系，决定了其功能的多样性。近年研究表明，伏隔核也参与疼痛的调节过程。胶质细胞在慢性疼痛产生和维持中的作用逐渐引起关注，胶质纤维酸性蛋白（glial fibrillary acidic protein ，GFAP）作为星型胶质细胞的骨架蛋白在其生物功能的执行中尤为重要。本研究旨在探讨NAc区星型胶质细胞GFAP是否参与慢性神经病理性疼痛的调节以及其可能的分子机制。.结果 1. 第一部分结果：.1.1慢性神经病理性疼痛小鼠CCI对侧NAc区星形胶质细胞特异性标记物GFAP在手术后7天、14天和21天表达增加（P<0.05）；.1.2 免疫荧光结果显示，CCI状态下，与假手术组和CCI同侧相比，CCI对侧NAc区星形胶质细胞明显增生活化；.1.3 免疫荧光显示，NAc区星形胶质细胞主要集中于core区。CCI14d，CCI对侧NAc core区注射星形胶质细胞抑制剂Fluorocitrate可明显缓解疼痛（P<0.01），而在CCI对侧shell注射则对PWL无影响；且在core或shell注射小胶质细胞抑制剂Minocycline均不能缓解疼痛行为；.1.4 CCI对侧NAc注射GFAP反义寡苷酸（ASO-GFAP）可使NAc区GFAP表达明显减少（P<0.001）；.1.5 CCI对侧NAc core注射ASO-GFAP可明显延长小鼠PWL（P<0.01）。.2. 第二部分结果：.2.1 免疫荧光结果显示，NAc区TNF-α主要与神经元（Neuron）共表达，与星形胶质细胞（Astrocyte）和小胶质细胞（Microglial）没有明显共标；.2.2 CCI14天小鼠NAc区TNF-α蛋白表达增加（P<0.01）；.2.3 CCI同侧NAc core区注射TNF-α，可明显缩短小鼠未造模侧PWL（P<0.05）；.2.4 CCI术后14天，CCI同侧NAc core区注射TNF-α，可诱发热痛觉过敏，再于NAc 区注射Fluorocitrate，可逆转TNF-α引起的疼痛行为（P<0.01）；.2.5 CCI术后14天，CCI同侧NAc core先注射ASO-GFAP干扰GFAP合成，24h后再注射TNF-α，TNF-α无法诱发疼痛行为（P<