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

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.

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。

“治痿独取阳明”是针灸疗法的一个重要取穴原则，但目前对于该取穴原则的理解存在争议，且相关具体作用机制尚不明确，影响治疗效果。已知表观遗传中的印迹基因Dlk1是肌肉萎缩和修复的关键。本研究探讨电针不同穴位对痿病疗效的影响，Dlk1基因在其中的可能作用及其上下游的调控机制。.研究以下肢失神经大鼠为痿病模型，以下肢行走功能、肌肉组织学、肌细胞凋亡和增殖分化为疗效指标，探讨电针治疗痿病的适宜的穴位组合。结果表明：①痿病模型大鼠下肢行走功能、肌湿重、肌细胞直径和横截面积降低，肌细胞凋亡水平升高，肌卫星细胞分化指标PAX7荧光染色强度增加；②针刺非经非穴位点上述指标均无改善；③针刺非阳明经穴位和阳明经穴位治疗可改善大鼠下肢行走功能、肌细胞直径和横截面积，并降低肌细胞凋亡指数，进一步促进肌卫星细胞分化；④针刺阳明经穴位治疗，肌湿重、肌细胞横截面积、肌细胞直径、肌细胞凋亡指数等指标的改善优于针刺非阳明经穴位，此外，还可以促进肌卫星细胞增殖指标PAX3荧光染色强度增加。即电针痿病肌肉局部的经穴可以改善大鼠痿病肢体功能和组织形态，降低肌细胞凋亡水平，促进肌细胞增殖分化，有效防治肌萎缩；电针阳明经穴肌萎缩改善程度明显优于非阳明经穴。.观察电针对萎缩肌肉细胞印迹基因Dlk1表达，及调控基因表达的表观遗传修饰水平和下游的可能作用信号通路的影响。结果表明：①电针可降低Dlk1基因的IG-DMR区的DNA甲基化和H3K27me3水平，但不改变H3K4me3、H3K9me2/3和组蛋白乙酰化水平；②上述表观遗传修饰水平的改变，使肌细胞内Dlk1的表达水平明显增高。③Dlk1蛋白浓度升高，对Notch信号通路的抑制作用增强，激活IGF1/PI3K/Akt信号通路，拮抗肌萎缩。基本明确了电针治疗痿病肌萎缩中以印迹基因Dlk1为核心的表观遗传调控机制的重要作用和其下游可能作用信号通路。.本研究为临床电针治疗痿病肌萎缩提供了穴位选择的科学依据；并初步阐明了电针防治痿病肌萎缩的以印迹基因Dlk1为核心的表观遗传调控机制，对电针治疗痿病具有理论探索和实践指导意义。