摘要目的:利用tFucci(CA)5细胞周期报告系统，观察不同细胞周期时相中细胞核、核仁与核质区室对电离辐射引起的DNA双链断裂(DSB)损伤响应及修复动力学。方法:通过共转染tFucci(CA)5和transposase表达质粒及单克隆细胞筛选，构建能稳定表达tFucci(CA)5的HT29细胞周期报告模型。利用2 Gy γ射线照射细胞，于照射后不同时间点经免疫荧光技术检测DSB标志物γ-H2AX foci，分析其在核仁区和核质区的形成与清除情况，探讨DSB损伤水平和修复动力学。结果:成功构建了稳定表达tFucci(CA)5的HT29细胞周期报告模型，实现了对G 1、S和G 2/M期细胞的精准识别与追踪。发现核仁区在放射诱导的DSB初始损伤中表现出更强的周期依赖性(G 1<S<G 2， F＝3.31~187.70， P<0.05)，与核质区的损伤模式存在差异(G 1 < S/G 2， F＝13.63~76.60， P<0.05)。DSB损伤修复动力学分析发现，在照射后中后期S期和G 2期细胞中核仁区DSB修复效率显著高于核质区( t＝2.41~8.55， P < 0.05)。 结论:本研究提供了可用于直观分析不同细胞周期时相DNA损伤情况的检测工具，发现细胞核仁与核质区室DSB损伤响应具有显著的细胞周期依赖性，在S期和G 2期细胞中核仁区DSB修复效率显著高于核质区。

abstractsObjective:To observe the responses of the nucleus, nucleolar compartment, and nucleoplasmic compartment of cells in different cycle phases to the DNA double-strand breaks (DSBs) induced by ionizing radiation and to analyze their DSB repair kinetics using the tFucci(CA)5 cell cycle reporter system.Methods:A HT29 cell cycle reporter model capable of stably expressing tFucci(CA)5 was constructed by co-transfecting the tFucci(CA)5 and transposase expression plasmids and selecting monoclonal cells. The cells were irradiated with 2 Gy of γ-rays. Post-irradiation, γ-H2AX foci, a DSB marker, were detected at different time points using an immunofluorescence technique. Accordingly, the formation and clearance of γ-H2AX foci in the nucleolar and nucleoplasmic compartments were analyzed to explore the damage levels and repair kinetics of DSBs.Results:The established HT29 cell cycle reporter model enabled the precise identification and tracking of cells in phases G 1, S, and G 2/M. The result indicated that to the initial DSBs induced by radiation, the responses of the nucleolar compartment exhibited greater dependence on cell cycle phases (G 1 < S < G 2, F = 3.31-187.70, P < 0.05), differing from the damage pattern of the nucleoplasmic compartment (G 1 < S/G 2, F = 13.63-76.60, P < 0.05). The analysis of DSB repair kinetics reveals that compared to the nucleoplasmic compartment, the nucleolar compartment of cells in the middle (S) and late (G 2) phases exhibited significantly higher DSB repair efficiency after irradiation ( t = 2.41-8.55, P < 0.05). Conclusions:This study provides a tool for the intuitive analysis of DNA damage in different cell cycle phases. The result reveal that the responses of the nucleolar and nucleoplasmic compartments to DSBs show significant dependence on the cell cycle. Furthermore, compared to the nucleoplasmic compartment, the nucleolar compartment of cells in the S and G 2 phases demonstrates significantly higher DSB repair efficiency.