NF-κB组织传感器的建立及其在检测低LET辐射诱导旁效应的运用

Ionizing radiation could not only directly damage the target cells, but also induce bystander effects in those non-targeted cells. Our understanding of radiation induced bystander effects is mostly based on the study of culture cells on monolayer, however, the aspects of molecular diffusion and communication between cells are completely different between cultured cells and cells in tissue, so it is desirable to study radiation induced bystander effects in tissue models. Our study on culture cells has shown that NF-κB could be activated by radiation induced bystander effects. In this study, we will use fluorescence energy transfer techniques to establish a tissue based biosensor of NF-κB activation, and then construct a 3D tissue model to study radiation induced bystander effects by implanting biosensor cells into mouse kidney tissue. By quantifying FRET efficiency, we will observe and study the temporal and spatial patterns of radiation bystander effects and illustrate if cytokines, TNF-α，IL-1 and IL-33 mediate the NF-κB activation caused by bystander effects. The study will observe radiation induced bystander effects and illustrate the molecular mechanisms of NF-κB activation in tissue level by establishing NF-κB biosensor. This study will provide us the experiment tools and theoretical basis for future study of radiation induced bystander effects in vivo.

电离辐射不仅能够直接损伤受辐射细胞，而且能够诱发那些未受辐射的细胞产生辐射旁效应。现在对于辐射旁效应的理解多基于细胞培养模型，但是由于培养细胞和组织的差异，我们亟需在组织模型中观察和研究辐射旁效应。申请人前期研究证实二维细胞培养中未受辐射的MCF-7细胞中NF-κB能通过辐射旁效应被激活。在此项目中，我们将NF-κB的活性变化作为检测辐射旁效应的一个指标，利用荧光共振能量转移（FRET）技术建立能够检测NF-κB激活的生物传感细胞，以此建立能够观察辐射旁效应的3D组织模型, 即融合了生物传感器的肾脏切片。通过量化FRET效率, 在肾脏切片中从空间和时间维度观察辐射旁效应，并阐述NF-κB在辐射旁效应中激活的分子机制。此项目通过NF-κB生物传感器的建立，达到在组织水平上观察辐射旁效应及阐述在辐射旁效应中NF-κB激活的机制，为将来在整体水平研究辐射旁效应提供实验工具和理论基础。

电离辐射不仅能够直接损伤受辐射细胞，而且能够诱发那些未受辐射的细胞产生辐射旁效应。我们的研究证明了放射线可诱导乳腺癌细胞MCF-7中NF-κB活性增高。活性增强的NF-κB通过介导TNF-α的大量分泌和参与端粒酶活性的调节等，最终引起癌细胞放射耐受增强，提高其生存优势进而引起肿瘤复发. (1)首次发现放射线不但可引起乳腺癌细胞中NF-κB的活性增高，并且被激活的NF-κB参与介导TNF-α的大量分泌，而TNF-α 也能通过自分泌激活被照射癌细胞的中NF-κB的活性和表达。NF-κB和TNF-α 可互相激活形成TNF-α - NF-κB - TNF-α 正反馈通路。(2) 放射线照射过的MCF-7细胞可通过TNF-α介导的辐射旁效应导致未照射细胞中的NF-κB活性增高。(3) NF-κB介导乳腺癌细胞的放射耐受性, 表现为放射线照射可通过NF-κB相关通路使MCF-7细胞具有更大的生存优势，阻断NF-κB活性则导致大量MCF-7细胞死亡；放射线照射过的MCF-7细胞可通过旁效应导致周围未照射癌细胞的端粒酶转录水平增加。本课题揭示NF-κB介导了肿瘤细胞的放射耐受的机制，从而为开发针对放疗后肿瘤复发的治疗提供理论基础。