基于印迹基因Dlk1探讨针刺阳明经穴防治痿病肌萎缩的表观遗传调控机制

项目名称： 基于印迹基因Dlk1探讨针刺阳明经穴防治痿病肌萎缩的表观遗传调控机制

项目编号： No.81503657

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 陈玄

作者单位： 福建省中医药研究院

项目金额： 18万元

中文摘要： 针刺阳明经穴防治痿病肌萎缩疗效确切，但作用机制不明，不利于针刺疗法发展。近研究表明，针刺可通过表观遗传调控产生疗效，另证据证明表观遗传中的印迹基因Dlk1是肌肉萎缩和修复的关键，故本研究提出以Dlk1为核心的表观遗传调控机制假说。拟观察坐骨神经损伤大鼠SFI、肌组织形态、肌细胞凋亡和肌卫星细胞增殖分化情况，从整体、组织和细胞水平初步阐明其生物学基础；进而分析Dlk1 mRNA和蛋白水平；并BSP检测Dlk1上游IG-DMR区甲基化，ChiP-PCR检测组蛋白H3、H4乙酰化和H3K4、H3K9、H3K27甲基化，阐明其上游表观调控作用；检测Notch和PI3K/AKT信号通路Notch、CSL、PI3K、AKT的mRNA和蛋白改变，阐明Dlk1下游标靶通路。从整体、组织、细胞水平和分子水平的Dlk1及其上下游通路的改变，层层深入揭示“治痿独取阳明”的表观遗传控机制，为针刺疗法提供理论依据。

中文关键词： 针刺研究；痿病；肌萎缩；表观遗传；印迹基因

英文摘要： The effect of acupuncturing the acupoints of yangming meridians to cure amyotrophy of flaccidity syndrome is proved. But the mechanism is unclear yet and it is disadvantaged to the application and promotion of acupuncture. On the basis of the development of the muscle regeneration and the preliminary work of our team, we suppose that it is regulated epigenetically with an imprint gene Delta-like 1 homolog (Dlk1) at the core. On the base of a rat model with sciatic nerve injury, five groups named control, model, acupuncturing the acupoints of yangming meridians, acupuncturing the acupoints except yangming meridians and acupuncturing the non-acupoints will be divided. Firstly, sciatic functional index (SFI) and histomorphyology will be detected. TUNEL and immunofluorescent staining methods will be used to observe the myocyte apoptosis and muscle satellite cells reproduction and differentiation to clarify the system, tissue and cellular mechanism of it. Secondly, realtime fluorogenic quantitative PCR (RTFQ PCR), Western-blot and ELLSA methods will be adopted to analyze the changes of the Dlk1 mRNA and protein content with acupuncture. And the molecular mechanism of DLk1 will be clarified. Thirdly, the DNA methylation level of the differentially methylated region (IG-DMR) and histone methylation and acetylation level upon Dlk1 will be detected by Bisulfite sequencing PCR (BSP) and chromatin immunoprecipitation assay (ChiP) technologies separately. The upstream epigenetically regulated mechanism of Dlk1 will be clarified after it. Finally, the mRNA and protein levels of Notch、CSL、PI3K、AKT which are key molecules in the NOTCH and PI3K/AKT signal pathways will be detected by RTFQ PCR and Western-blot methods. The downstream signal pathway of Dlk1 during muscle regeneration will be clarified. The study probes Dlk1 celluar and molecule mechanism of acupuncturing the acupoints of yangming meridians to cure amyotrophy of flaccidity syndrome and the upstream and downstream effect to illuminate the mechanism systematically. It will lay a foundation for selecting the acupoints and operating technique better in clinical and provide a helpful reference to the other studies of acupuncture mechanism.

英文关键词： acupuncture research;flaccidity syndrome;amtrophy;epigenetics;imprinting gene