γ-H2AX分析电离辐射诱发小鼠神经元DNA双链断裂及修复

Ionizing radiation-induced DNA double-strand break and repair assessed by γ-H2AX foci analysis in neurons in mice

目的观察临床剂量的电离辐射后小鼠神经元DNA双链的断裂及修复，探讨γ-H2AX是否能作为衡量体内正常脑组织神经元DNA双链断裂（DSB）形成和修复的指标。方法电离辐射诱导DSB形成试验，C57BL／6小鼠行0．1、0．5和1．0Gy全身照射后10min，收集脑组织进行分析；DSB修复试验，修复功能正常小鼠（C57BL／6）和修复缺陷鼠（BALB／c，A—T和SCID）在全身照射2Gy后0．5、2．5、5、24和48h收集脑组织进行分析。未照射的小鼠作为对照组。γ-H2AX和NeuN免疫荧光双重染色和免疫组织化学染色分析脑组织神经元DSB形成和修复。结果DSB形成试验，在对照组脑组织皮质区神经元的细胞核内仅有数目很少的γ-H2AX焦点，而受照射后细胞核内γ-H2AX焦点数目显著增加，并显示出明显的剂量相关。通过分析不同放射敏感性的小鼠电离辐射后脑组织皮质区神经元的DSB修复动力学，发现C57BL／6小鼠细胞核内γ-H2AX焦点随时间的延长迅速减少，在照射后24和48h仅有很低水平DSB未修复；而免疫缺陷SCID鼠在照射后所有的时间点都显示出γ-H2AX焦点的明显增加，A—T小鼠表现出较低的修复缺陷，主要表现在较晚的时间点（≥5h）γ-H2AX焦点的中度增加；放射敏感的BALB／c小鼠与C57BL／6小鼠相比γ-H2AX焦点数量轻度增加。结论γ-H2AX焦点分析可以作为一项精确的量化指标，在体内衡量临床相关剂量电离辐射诱导的DSB形成和修复。

Objective To investigate if the γ-H2AX loci is a precise index for the DSB formation and repair in mature neurons of brain in vivo after clinically relevant doses irradiation. Methods For the DSB formation experiment, the mature neurons in the neocortex of brain tissue of C57BL/6 mice were analyzed at 10 min after whole-body irradiation with 0. 1, 0.5 and 1.0 Gy. For the DSB repair kinetics experiment, the mature neurons in the neocortex of brain tissue of repair-proficient （C57BL/6 mice） and repair-deficient mouse strains （BALB/c, A-T and SCID mice） were analyzed at 0.5, 2.5, 5, 24 and 48 h after whole-body irradiation with 2 Gy. The mature neurons in the neocortex of brain tissue of sham-irradiated mice of each strain served as controls. γ-H2AX immunohistoehemistry and γ-H2AX and NeuN double immunofluoreseence analysis was used to measure DSBs formation and repair in the mature neurons in the neocortex of brain tissue of the different mouse strains. Results For the DSB formation experiment, γ-H2AX foci levels with a clear linear dose correlation and very low backgrounds in the nuclei in the neocortex of brain tissue were observed. Scoring the loss of γ-H2AX loci allowed us to verify the different, genetically determined DSB repair deficiencies, including the minor impairment of BALB/e mice. Repair-proficient C57BL/6 mice exhibited the fastest decrease in loci number with time, and displayed low levels of residual damage at 24 h and 48 h post-irradiation. In contrast, SCID mice showed highly increased γ-H2AX foci levels at all repair times （0.5 h to 48 h） while A-T mice exhibited a lesser defect which was most significant at later repair times （ ≥ 5 h）. Radiosensitive BALB/c mice exhibited slightly elevated foci numbers compared with C57BL/6 mice at 5 h and 24 h but not at 48 h post-irradiation. Conclusion Quantifying the γ-H2AX foci in normal tissue represents a sensitive tool for the detection of induction and repair of radiation-induced DSBs at clinically relevant doses in vivo.