γ-H2AX分析用于检测电离辐射致HepG2细胞DNA双链断裂的研究

γ-H2AX assay as a measurement of DNA double-strand breaks in irradiated HepG2 cells

目的 应用γ-H2AX分析检测电离辐射导致肝癌细胞株HepG2基因组DNA双链断裂的情况，并检测在不同细胞周期时相中的表达差异，从而了解不同时相HepG2细胞株的辐射敏感性。方法利用胸腺嘧啶核苷（TdR）阻断HepG2细胞于G1期末，于不同时间点分别得到同步化的S期和G2/M期细胞，用60Coγ射线照射细胞，建立DNA双链断裂模型，采用免疫荧光法和Westemblotting检测γ-H2AX的表达。结果TdR阻断后继续培养至34和40h，分别得到了较高同步化程度的S期和G2／M期HepG2细胞；受照后的HepG2细胞中Y-H2AX表达较照射前显著增高，处于S期的细胞γ-H2AX表达增加更为突出。结论不同周期时相的HepG2细胞受照后均检测出不同程度的DNA双链断裂，其中S期细胞尤为敏感。γ-H2AX对DNA双链断裂快速敏感的反应使γ-H2AX分析在检测早期DNA双链损伤中具有广泛的廊用前景。

Objective To explore the possibility of detecting DNA double-strand breaks （DSBs） in irradiated HepG2 cells by γ-H2AX assay and to investigate the γ-H2AX expression in different cell cycle phases. Methods HepG2 cells were blocked by TdR in G1 phase of cell cycle, and then cultured for different hours after TdR was removed. Synchronous S phase cells and G2/M phase cells were collected respectively and irradiated with 60Coγ rays. Western blotting and immunofluorescence were used to analyze the expression of γ-H2AX in cells. Results Synchronous S phase cells and G2/M phase cells were obtained at hours 34 and 40 post TdR block respectively. γ-H2AX level increased rapidly after cells were exposed to 60Coγ rays, especially in S phase of the cell cycle. Conclusions The cells in S phase are more prone to be damaged by ionizing radiation than those in G2/M. γ-H2AX assay can be regarded as an early diagnosis indicator of DSBs.