肥大细胞源性Exosome介导针刺穴位神经-免疫调节启动的初步研究

The 2013 Nobel Prize in Physiology or Medicine was awarded to the discovery of transportation and regulatory mechanism of vesicle. Exosome, the common form of vesicle transportation between cells, has important effect on immunological regulation. The existing researches at home and abroad and the preliminary work of our research group have shown that the produce of acupuncture effects is closely related with the nerve, mast cell in the local region of acupoints. Mast cell-derived exosome(MC-Exo）,can activate adjacent sensory nerve, promote nerve terminal releasing P substance and so on. Based on the close relationship of exosome with nerve and mast cell, we hold that exosome, released from mast cell by needling acupoints, plays vital role in the nerve-immune regulation initiating by needling acupoints and acts as one of the key factors of acupuncture effect initiating. This project applies acupuncture and moxibustion, molecular biology, neurophysiology and bioinformatics comprehensively , studies the influence of acupuncture on MC-Exo expression in acupoints ,observes the function of MC-Exo in nerve-immune regulation by needling , probes the biological mechanism of nerve-immune regulation initiating mediated by Mast cell-derived Exosome needling acupoints and provides a basis for the study of initiate of acupuncture effect.

2013年诺贝尔生理学或医学奖授予细胞囊泡运输调控机制的重大发现，外来体（Exosome）是细胞间囊泡运输的常见形式，具有重要的免疫调节作用。国内外现有研究和本课题组前期工作表明，针刺效应的产生与穴位局部神经和肥大细胞关系密切。肥大细胞源性Exosome（MC-Exo）可激活邻近感觉神经兴奋，促进神经末梢释放P物质等。基于Exosome与神经、肥大细胞的密切关系，我们认为针刺穴位引起肥大细胞释放的Exosome在针刺穴位局部神经-免疫调节启动的过程中具有重要作用，是针刺起效的关键因素之一。本项目综合采用中医针灸学、分子生物学、神经生理学、生物信息学等技术与方法，研究针刺对穴位局部MC-Exo表达的影响，观察MC-Exo在针刺神经-免疫调节效应中的作用，探索MC-Exo介导针刺穴位启动神经-免疫调节的部分生物学机制，为针刺穴位效应启动机制提供实验依据。

Exosome是可以携带蛋白质，脂质，核酸等生物信息的细胞外囊泡，是细胞网络通讯的重要载体。针刺作为一种物理刺激，网络调节是针刺作用的基本途径。本项目在电针治疗佐剂性关节炎镇痛抗炎效应研究平台上，综合采用Western blot、ELISA，label free蛋白组学等技术及生物信息学分析方法，观察针刺对正常和RA模型大鼠血清Exosome 含量的影响；探讨穴位局部Exosome在针刺治疗起效中的作用；探索Exosome介导针刺神经免疫调节的作用及机制。结果发现1.针刺可以促进正常大鼠血清Exosome的释放；佐剂性关节炎病理状态下，疾病早期，针刺可抑制血清Exosome 的释放；疾病后期，针刺可以促进大鼠血清Exosome 的释放.2.拮抗穴区局部exosome可以显著减弱针刺镇痛效应.3.大鼠血清exosome内含物质的蛋白组学分析，发现电针可抑制由造模导致的12个差异显著的促炎因子的释放，促进8个差异显著的抑炎因子的释放。与模型组相比，针刺组出现了22个新生蛋白，而这些蛋白多与神经免疫调节关系密切。根据复杂网络分析结合生物学意义，最终确定了4个针刺关键蛋白，它们是补体C3（C3），蛋白Cfh和C4b结合蛋白α链以及补体C1q/肿瘤坏死因子相关蛋白3（CTRP3），这些关键蛋白可能在针刺神经免疫调节中起到关键节点作用。本项目首次从血清exosome角度阐释了针刺神经免疫调节启动的部分生物学机制。