X射线辐照诱导人神经胶质瘤细胞DNA双链损伤研究

采用免疫荧光染色法检测X射线诱导M059K细胞磷酸化组蛋白H2AX(γH2AX)和磷酸化的毛细血管扩张性共济失调症突变基因(pATM)焦点的形成;流式细胞仪分析M059K细胞γH2AX和pATM表达的周期依赖性。免疫荧光染色检测结果显示,γH2AX和pATM焦点阳性细胞分别以照射后0.5、4 h表达最高,照射后24h,高剂量组焦点阳性细胞达100%。流式细胞仪分析结果显示,γH2AX和pATM的表达具有细胞周期依赖性。照射后0.5、4 h,γH2AX和pATM在细胞各期的表达均明显增加,并以G0/G1期为著;照射后24 h,γH2AX和pATM在G0/G1和S期表达降低,而G2/M期细胞表达明显增加。细胞呈现明显的G2/M期阻滞。结果提示,γH2AX和ATM信号通路参与X射线诱导的M059K细胞DNA双链断裂。

DNA double-strand breaks induced by X-ray irradiation in human glioma cells

The foci of phosphorylated histone H2AX (γH2AX) and phosphorylated ataxia telangiectasia mutated (pATM) were visualized using immunocytochemical methods. Flow cytometry was used to assay γH2AX and pATM expression and the cell cycle. The results of immunocytochemical assay showed that the strongest induction of γH2AX and pATM foci appeared at 0.5 h and 4 h after irradiation, and the positivity for γH2AX and pATM foci reached 100% at 24 h with X-ray exposure in higher-dose groups. Flow cytometry results showed that the expression of γH2AX and pATM was related to the cell cycle. The rise of γH2AX and pATM expression was shown in each phase at 0.5 h and 4 h post-exposure, and this increase was more obvious in G0/G1 phase. At 24 h post-exposure, the expression of γH2AX and pATM decreased in G0/G1 and S phase, while increased significantly in G2/M phase. There was significant prevalence of the arrest at the G2–M transition among M059K cells. The above results indicate that the signaling pathways of γH2AX and pATM is involved in X-ray-induced double-strand breaks in M059K cells.