CAJ | 학술논문

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、O.5和1.0 Gy全身照射后10 win,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2 Gy后0.5、2.5、5、24和48 h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48 h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(≥5 h)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.
목적 관찰림상제량적전리복사후소서신경원DNA쌍련적단렬급수복,탐토γ-H2AX시부능작위형량체내정상뇌조직신경원DNA쌍련단렬(DSB)형성화수복적지표.방법 전리복사유도DSB형성시험,C57BL/6소서행0.1、O.5화1.0 Gy전신조사후10 win,수집뇌조직진행분석;DSB수복시험,수복공능정상소서(C57BL/6)화수복결함서(BALB/c,A-T화SCID)재전신조사2 Gy후0.5、2.5、5、24화48 h수집뇌조직진행분석.미조사적소서작위대조조.γ-H2AX화NeuN면역형광쌍중염색화면역조직화학염색분석뇌조직신경원DSB형성화수복.결과 DSB형성시험,재대조조뇌조직피질구신경원적세포핵내부유수목흔소적γ-H2AX초점,이수조사후세포핵내γ-H2AX초점수목현저증가,병현시출명현적제량상관.통과분석불동방사민감성적소서전리복사후뇌조직피질구신경원적DSB수복동역학,발현C57BL/6소서세포핵내γ-H2AX초점수시간적연장신속감소,재조사후24화48 h부유흔저수평DSB미수복;이면역결함SCID서재조사후소유적시간점도현시출γ-H2AX초점적명현증가,A-T소서표현출교저적수복결함.주요표현재교만적시간점(≥5 h)γ-H2AX초점적중도증가;방사민감적BALB/c소서여C57BL/6소서상비γ-H2AX초점수량경도증가.결론 γ-H2AX초점분석가이작위일항정학적양화지표,재체내형량림상상관제량전리복사유도적DSB형성화수복.
Objective To investigate ff the γ-H12AX foci 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 rain 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 immunohistochemistry and γ-H2AX and NeuN double immunofluorescence 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-H12AX foci allowed us to verify the different, genetically determined DSB repair deficiencies, including the minor impairment of BALB/c mice. Repair-proficient C57BL/6 mice exhibited the fastest decrease in foei 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 slighdy elevated foei numbers compared with C57BI./6 mice at 5 h and 24 h but not at 48 h post-irradiation. Conclusion Quantifying the γ-H2AX loci in normal tissue represents a sensitive tool for the detection of induction and repair of radiation-induced DSBs at clinically relevant doses in vivo.