microRNA155靶向P2X7介导艾灸足三里穴位皮肤免疫激活的表观遗传调控机制

Based on novel findings in our previous studies that both the skin immunity on acupoint would get involved in the mechanism of moxibustion therapy and P2X7 would be a key molecule at local acupoint responding to moxibustion , and combining the fact that the relationship between miR-155 and P2X7 which play important role in immune system network regulation, we propose that “microRNA155 targeting P2X7 activating local skin immune on acupoint would be able to the key initiating step of moxibustion effect”. Guided by this hypothesis, we will give moxibustion stimulation at ST36 ,establish adjuvant arthritis mice model, and apply flow cytometry, immunofluorescence multi-label techniques, miRNA research's classic technology (real-time fluorescent quantitative PCR, mimics excited, inhibitor suppression, dual luciferase miRNA target validation), conditional knockout of P2X7 receptor, and so on, to reveal the role of microRNA155-P2X7 in local skin immune activation of moxibustioned acupoints and expect to provide more evidences to underlying the initiating mechanism of moxibustion at the local acupoint.

基于皮肤免疫激活与艾灸始动机制的密切关系，结合microRNA是表观遗传调节子，且microRNA155与P2X7的靶向调控在皮肤免疫激活中的重要作用，我们提出“microRNA155靶向P2X7的表观遗传调控是艾灸激活穴位皮肤免疫的关键分子机制”工作假说，并拟以足三里为穴位代表，以佐剂性关节炎动物模型为研究载体，以“microRNA155靶向P2X7的表观遗传调控”为切入点，综合运用流式细胞分选、免疫荧光多标、microRNA研究的经典技术（实时荧光定量PCR、mimics激动、inhibitor抑制、双荧光素酶miRNA靶点验证）、P2X7条件基因敲除，从正、反两个途径，揭示microRNA155-P2X7表观遗传调控在艾灸激活穴位皮肤免疫中的作用机制，为深入阐释艾灸作用的穴位局部始动机制，推动艾灸作用原理的深入研究提供丰富的科学依据。

本项目紧密围绕“microRNA155靶向P2X7的表观遗传调控是艾灸激活穴位皮肤免疫的关键分子机制”工作假说，以CFA诱导的佐剂型关节炎为研究载体，综合运用病理形态学、ELISA、免疫荧光多标、双光子显微成像、microRNA经典研究技术（Q-PCR、表达谱测序、基于CRISPR-Cas9构建miR155条件基因敲除鼠、agomir激动、antagomir抑制、双荧光素酶miRNA靶点验证）、P2X7基因敲除等技术，分别开展了①艾灸治疗佐剂性关节炎疗效、②艾灸足三里穴位局部皮肤免疫激活效应、③艾灸足三里穴位局部皮肤免疫激活的miR-155参与机制、④艾灸足三里穴位局部皮肤免疫激活的P2X7参与机制、⑤艾灸足三里穴位局部皮肤免疫激活的microRNA155-P2X7靶向表观遗传调控机制的研究，研究发现:艾灸后穴位局部可见树突细胞激活、募集和迁移；多个microRNA参与艾灸后穴位皮肤免疫激活，miR-155是其关键调节分子之一;miR-155-3p和miR-155-5p均参与艾灸后穴位皮肤免疫激活；microRNA155靶向P2X7调控穴位皮肤免疫激活可能是艾灸效应启动的重要环节。该研究从免疫调节的角度探索了艾灸穴位局部始动机制，为艾灸作用的科学原理的揭示提供了实验依据，也为“针所不为，灸之所宜“的科学内涵阐释提供了一些线索。发表课题研究相关SCI论文7篇，2篇待发；培养已毕业硕博研究生各1名，在读硕博研究生5名。