针刺山羊中枢基因表达谱分析及镇痛相关神经肽调控作用研究

Acupuncture analgesia is not only an important surgical technique, but also one of the important research areas in neuroscience. As acupuncture effect is involved in a variety of neural and humoral factors, its mechanism is not elucidated yet. Studies show that acupuncture modifies analgesia through the release of neuropeptides and their gene expression. Since numerous kinds of neuropeptides are found in the central nerve system, their relationship with acupuncture analgesia needs to be studied. Acupuncture induces better analgesic effect in ruminants than in human or other animals, so there may exist specific molecular regulatory mechanism by which acupuncture induces analgesia in ruminants. In this study, goats with the same genetic background are selected for paired experiment. The trial goats receive acupuncture for 30 min. Samples of periaqueductal grey, parabrachial neuclei and arcuate neucleus of the central nerve system are taken respectively 4 min after the acupuncture. Deep sequencing of transcriptomes is conducted with Seloxa technology. Differential gene expression profile is analyzed with bioinformatics methods to determine analgesia-related genes. On this basis, genes of related neuropeptides are selected to clone and express, and antibodies to the neuropeptides are prepared. Patterns of the distribution and acupuncture-induced release of the neuropeptides are determined with the immunohistochemical strept avidin-biotin-peroxidase complex staining technique. The neuropeptides or corresponding antibodies or antisence RNAs to their genes are microinjected into the specific cerebral neuclei in goats to determine roles of the peptides in acupuncture-induced analgesia. This study is conducive to completely elucidate the regulating mechanisms by which acupuncture induces analgesia, and to promote the development of neuroscience and the application of acupuncture technique in veterinary clinical practice.

针刺镇痛是重要的外科技术，是神经科学领域的重要研究方向之一。针刺镇痛效应涉及中枢神经体液众多因素，其机理尚未完全阐明。研究表明针刺通过诱导神经肽基因表达和神经肽释放参与镇痛调节作用。因神经肽种类繁多，其与针刺镇痛的关系有待研究。反刍动物针刺镇痛效果优于人和其它种属动物，因此反刍动物针刺镇痛还存在特定的分子机制。本项目使用遗传背景相同山羊做针刺和非针刺配对实验，针刺30min，4h后分别取中脑导水管周围灰质、臂旁核和弓状核样品，Solexa法深度测序，生物信息学方法分析基因差异表达谱，确定针刺镇痛相关基因。选取相关神经肽基因克隆、表达，表达产物纯化、制备抗体。采用SABC法确定神经肽的中枢分布及针刺诱导的释放规律，核团微注射神经肽及其抗体和反义mRNA分别至山羊特定核团，确定其在针刺镇痛中的调控作用。该研究有助于全面揭示针刺镇痛的调节机制，促进神经科学的发展和针刺镇痛技术在兽医临床上的应用。

针刺镇痛是神经科学领域的重要研究方向之一。据研究报道针刺可诱导动物中枢多种镇痛相关基因在不同神经核团表达。中枢已知神经肽有100多种，但绝大多数与针刺镇痛效应的关系尚未有研究。本研究通过山羊转录组测序建立针刺镇痛分子机制研究平台，筛选出2000多个显著表达差异基因，通过GO富集分析获得了参与针刺调节90个GO，KEGG Pathway分析获得5条电针相关的有研究价值的通路。从验证的基因中选取胸腺素β4基因、兴奋性氨基酸转运体基因、阿片肽基因、c-fos 和 c-fun，进行了相关功能的研究。侧脑室注射胸腺素β4，发现在生理和病理情况下胸腺素β4剂量依赖性拮抗针刺镇痛作用；电针增加胸腺素β4在中枢神经系统多个镇痛相关核团表达，提示胸腺素β4参与了电针诱导的中枢神经保护机制。本试验建立了病理痛模型和耐受模型，鞘内注射兴奋性氨基酸转运体（EAATs）拮抗剂，检测脊髓中EAATs表达的变化，证明EAATs参与电针治疗神经病理痛和针刺耐受。电针诱导山羊脑啡肽前原、前阿黑皮素、强啡肽前原及其δ-、 µ-和 κ-受体表达增加，其增加水平与痛阈成正相关，提示阿片肽及其受体可能与电针诱导的镇痛后效应有关。本项目对下丘脑MIRNA进行深度测序，并鞘内注射拮抗剂或激动剂，初步确定miR-148a-3p、miR-370-3p、let-7b-5p、miR-107-3p以及miR-124-5p参与电针耐受。采用不同频率电针刺激山羊，研究了中枢c-Fos和 c-Jun表达水平和痛阈之间的关系，发现60 Hz电针镇痛效果优于0、2和100 Hz，电针诱导c-Fos和c-Jun在多数镇痛相关核团中表达增加；通过比较电针不同穴位的镇痛效果以及c-Fos在镇痛相关核团的表达情况，发现电针不同穴位产生镇痛的效果有差异，且能激活共同的神经通路：弓状核-中脑导水管周围灰质-中缝大核/蓝斑-脊髓和下丘脑-垂体。. 本实验按照项目计划完成，发表了高质量SCI论文7篇，学位论文5篇。建立了针刺镇痛分子机制研究平台，为针刺镇痛机制的深入研究和其临床应用奠定了基础。