DNA-PKcs-Chk2参与细胞周期相关辐射旁效应的机制研究

Radiation-induced bystander effect (RIBE) comprises dual functions including sensitization and adverse reaction, and this "double-edged sword" role is a key factor that influences the curative effect of cancer radiotherapy. Our preliminary research demonstrated that DNA-PKcs-Chk2 regulated mitosis progression and phosphorylation of DNA damage response protein in mitosis. In addition, pilot studies indicated that RIBE could occur in cells at different cell cycle status in response to various LET radiation, but the underlying mechanisms remain unclear. Therefore, we hypothesized that DNA-PKcs-Chk2 and their downstream factors may regulate cell cycle-related RIBE. To validate our hypothesis, based on our preliminary results, we would like to elucidate the possible mechanisms of the involvement of DNA-PKcs-Chk2 in regulating cell cycle-related RIBE from molecular, cellular and animal levels, providing scientific basis into cancer radiotherapy strategies and excavating potential therapeutic targets.

辐射旁效应在放疗过程中存在增敏和毒副反应的双重作用，其扮演的“双刃剑”角色是影响放疗疗效的关键因素。我们前期研究证实，DNA-PKcs-Chk2调控细胞有丝分裂进程和相关DNA损伤反应蛋白在有丝分裂期的磷酸化，另外，预实验结果显示，不同LET射线照射均可引起处于不同周期状态的细胞产生辐射旁效应，且DNA-PKcs参与细胞周期相关的辐射旁效应，但其具体机制尚不清楚。为此我们提出假设，细胞周期和辐射旁效应密切相关，且很可能受DNA-PKcs-Chk2及其下游通路的调节。为了验证这一假设，本课题拟在前期研究的基础上，围绕“DNA-PKcs-Chk2-细胞周期相关辐射旁效应-增敏和毒副反应”这一主线，从分子，细胞及动物等多方面进行深入探讨，阐明DNA-PKcs-Chk2参与细胞周期相关辐射旁效应的机制，进而为肿瘤放疗策略的制定及挖掘新的潜在治疗靶点提供依据。

电离辐射旁效应在肿瘤放射治疗中扮演着“双刃剑”的角色，一方面可以提高肿瘤细胞辐射敏感性，另一方面又会对正常细胞产生毒副作用。本项目以DNA-PKcs缺失的正常细胞模型及DNA-PKcs敲低的肿瘤细胞模型为研究对象，利用不同类型的射线进行电离辐射旁效应的研究。采用条件培养液处理法处理旁细胞；TDR双阻断释放法及Nocodazole处理法进行细胞周期同步化；微核形成实验检测细胞DNA损伤；克隆形成实验检测细胞存活；流式细胞术检测细胞周期；western blot检测蛋白表达；荧光探针法测定ROS水平等。当前研究主要发现在DNA-PKcs缺失的细胞模型中观察到靶细胞中DNA-PKcs的缺失会导致不同类型电离辐射诱导正常细胞的旁效应被抑制，而旁细胞中DNA-PKcs的缺失增强了旁细胞的损伤应答。当在有丝分裂期细胞中敲低DNA-PKcs，会导致ROS表达水平下降；敲低DNA-PKcs或抑制ROS后，发现有丝分裂期相关辐射旁效应显著降低，且同时敲低DNA-PKcs和抑制ROS后，观察到有丝分裂期相关辐射旁效应进一步降低，提示DNA-PKcs/ROS信号轴参与调控有丝分裂期相关辐射旁效应。通过本项目的研究，有利于加深对DNA-PKcs参与的电离辐射旁效应影响到肿瘤细胞放疗增敏及正常细胞毒副作用的认识，为肿瘤精准放疗计划的实施及放疗增敏和防护新靶点的发现提供思路和依据。