外泌体在辐射远端效应中的作用及其分子机制研究

Local body irradiation is popular in radiation accident and radiotherapy, and the radiation harmful effect is mainly depended on the damage situation of radiosensitive tissues. Based on our previous plenty works on cell radiation responses, this program will study the mechanisms of radiation induced abscopal effect (RIAE) by locally irradiating the right lung of mouse with X-rays and investigate the roles of exosomes in this effect by treating the mouse with an inhibitor of exosome release before and after irradiation. After 1d to 6 months of irradiation, the cell damage and pathological alters in the left lung, small intestine and its stem cells , marrow and mesenchymal stem cells (MSC), and blood will be measured. The exosomes in the blood before and after irradiation will be collected to assay the containing proteins and miRNAs and the changes will be analyzed. After being marked with a stable fluorescence dye, the exosomes will be injected into mouse though caudal vein and its distribution in the body will be detected. In addition, the cell and tissue responses to the external exosomes will be measured. Accordingly to the obvious changes of the proteins and miRNAs in the exosomes before and after irradiation, their over-expression plasmids and siRNAs will be constructed and transferred into a pulmonary epithelial cell MLE-12. After irradiation, the exosomes from irradiated MLE-12 cells are collected and send to MSC and small intestinal crypt epithelial cell IEC-6 in order to identify the functional proteins and miRNAs. The molecule targets of miRNAs will be studied as well. Moreover, the agomir and antagomir of a few selected miRNAs will be injected into mouse to further verify the roles of miRNAs in RIAE. The new signal factors of RIAE can be expected from this study, which could provide new treatment strategies for radiation injury.

局部照射是核事故和放疗普遍存在的受辐射方式，敏感组织损伤是辐射危害的内因。本项目拟在我们前期大量细胞水平研究积累的基础上，从个体水平上研究辐射远端效应以及外泌体在其中的作用。以X射线定位照射小鼠右肺后1天至6个月，检测小鼠左肺、小肠、骨髓、血液等组织的病理变化和其中干细胞的损伤，通过外泌体抑制剂确定外泌体在辐射远端效应中的作用；收集辐照前后小鼠外周血中外泌体，分析其中蛋白和miRNA分子的变化，检测辐射诱导的外泌体在体内的分布及其对骨髓、小肠及它们所含干细胞的损伤；基于辐射前后外泌体中显著差异蛋白和miRNA，建立相应的过表达载体和抑制基因并将之转染肺上皮细胞，研究受辐射肺细胞所释放外泌体对骨髓干细胞、小肠隐窝细胞等的损伤；通过构建miRNA分子模拟物和动物实验验证，获知几种与辐射远端效应密切相关的miRNAs，探索其分子靶点，有望发现新的辐射信号分子，为放射损伤救治与防护提高新的策略。

随着核技术的广泛应用，辐射对健康的危害效应一直受到人们的重视。局部照射是核事故和放疗普遍存在的受辐射方式，照射野以外敏感组织器官的远后效应是近年来研究的热点。本项目利用射线照射小鼠或大鼠的右侧胸部，发现出可引起动物血象学的变化外，还可以引起未受照射左肺的炎症反应甚至纤维化，对远端的股骨骨髓细胞、生殖系统产生损伤效应。深入研究发现，胸部照射后，小鼠睾丸血睾屏障被破坏，胞间链接蛋白水平下降，精子细胞发生凋亡，活力下降，并产生子代存活率下降等遗传效应，且受辐射小鼠的血清具有细胞毒性。通过对血清蛋白质芯片检测、组学检测与生物信息分析、激素水平检测等，发现炎症因子在该辐射远端效应中发挥重要作用，而抗炎药物西米替丁可以有效降低辐射远端效应。同时，利用细胞共培养模型实验发现，巨噬细胞可以通过应激释放IL-8、TNF-等炎症因子加重受辐射肺细胞的致死性损伤，介导不同射线照射诱发的p53依赖的二次旁效应，而自噬可以对细胞具有辐射保护作用。研究发现，受辐射细胞可以释放大量外泌体，且随辐照剂量的增大和辐照后时间的延长而增加，促进周围细胞侵袭转移能力，降低受体细胞的辐射敏感性；通过基因芯片与组学检测、功能验证，发现外泌体ALDOA和ALDH3A1蛋白通过加速糖酵解促进细胞迁移，而受辐射肺细胞所释放的miRNA-1246则以非外泌体携带方式存在于胞外，通过作用于受体细胞的DR5靶点，促进旁细胞增殖，增强其辐射抗性。外泌体常包含细胞器成份，发现内质网应激反应可通过激活PERK–p-eIF2α等信号通路抵御辐射旁效应损伤，线粒体ROS则是引起辐射旁效应的重要因素之一。本项目所发现的辐射远端效应对子代产生遗传影响，对放射治疗的生物安全提出了新的挑战，具有创新科学意义；通过在线粒体、内质网等亚细胞结构水平研究辐射旁效应，诠释了新的理论；通过挖掘介导辐射旁效应的外泌体蛋白和miRNA，为进一步掌握外泌体中关键旁效应信号分子研究打下了良好的实验基础。