天然免疫信号分子MAVS介导辐射旁效应及机理研究

The mitochondrion is “the powerhouse of the cell” to supply cellular energy and plays important roles in the ionizing radiation induced lesions in cells. MAVS, a mitochondrial antiviral signaling adaptor, is critical for host defenses against viral infection by activating the transcription factors of IRF3/7 and NF-B, which lead to the expression of interferon to activate antiviral innate immunity. As an signaling adaptor with antiviral feature in the mitochondrial membrane, whether MAVS respond to ionizing radiation and how it affects on the radiosensitivity remain unclear. Our preliminary work showed that radiation with X-rays affected the expression of MAVS in cells. Knockdown of MAVS in donor cells could modulate radiation-induced bystander effect and protect the bystander cells of high-expression MAVS. In the proposed project, we are going to knock down MAVS by siRNA transfection or overexpress MAVS by plasmid transfection in cells and then the cells are irradiated by X-rays and carbon ions beam. The formation of micronucles, DSB repair protein foci and apoptosis, et al., will be investigated to assess the bystander effect after the MAVS expression modification and its potential application in combination with radiotherapy. Further, the related signaling pathway involving in MAVS will be investigated by high through sequence of the exosome, ELISA，and western blot to indicate the mechanisms of the immune molecule response to bystander effect. The general aim of the project is to provide reference for the designs of radiation protection and clinical radiotherapy.

线粒体是细胞能量代谢的中心，在电离辐射诱导的损伤效应中地位重要。MAVS蛋白是线粒体膜中发现的第一个与天然免疫相关的蛋白，通过活化IRF3/7及NF-B等信号通路，进而激活干扰素表达，诱发细胞内抗感染天然免疫反应。作为线粒体膜表面蛋白并具有先天免疫信号功能的MAVS，是否响应辐射应答？其对辐射旁效应的影响又是怎样？目前尚不清楚。初步研究显示，细胞中MAVS表达响应X射线辐照；敲低供体细胞MAVS后辐照处理，保护MAVS高表达的旁细胞。为深入研究其机理、探讨与临床放疗结合应用的可能性，本项目拟采用MAVS载体超表达和siRNA沉默相关细胞，X射线与碳离子辐照后与受体细胞共培养检测细胞微核、DSB修复蛋白等生物学指标，分析MAVS介导的辐射旁效应；采用外泌体高通量测序、ELISA、WB分析等方法检测分析其调控机制，揭示免疫分子MAVS参与辐射旁效应及机理，为辐射防护与临床放疗提供参考依据。

线粒体是细胞能量代谢的中心，在电离辐射诱导的损伤效应中地位重要。MAVS蛋白是线粒体膜中发现的第一个与天然免疫相关的蛋白，通过活化IRF3/7及NF-B等信号通路，进而激活干扰素表达，诱发细胞内抗感染天然免疫反应。我们研究表明，敲除MAVS可减轻辐射诱导的线粒体膜电位破坏和ATP产生等线粒体功能障碍，可下调促凋亡蛋白的表达降低凋亡水平，同时可减少辐射后细胞内ROS的产生。此外，与对照组相比，线粒体靶向抗氧化剂MitoQ抑制线粒体ROS可降低辐照后MAVS寡聚化的数量，并可预防微核的发生，提高辐照后正常A549细胞的存活率。细胞共培养实验表明，MAVS参与了辐射引起的旁效应（RIBE）。有趣的是，在MAVS表达水平较高的受体细胞A549中，RIBE反应比MAVS水平较低的受体细胞BEAS-2B更为显著。我们的研究表明，先天免疫信号分子MAVS通过辐射诱导ROS介导的寡聚化参与辐射效应，从而导致mtROS的增加，进而诱导更多的MAVS寡聚化，扩大辐射诱导的损伤。ROS也是通过MAVS通路参与RIBE的重要因子，我们的研究提示MAVS可能是一个潜在的精确放疗和放射防护靶点。