摘要
目的研究S期细胞热损伤后DNA双链断裂形成规律,以阐明S期细胞热敏感原因,并分析DNA双链断裂延迟性形成的可能机制。方法流式细胞术分析热损伤后细胞周期阻滞;Ed U掺入实验检测DNA复制能力;血清饥饿法同步H1299细胞周期;中性彗星实验动态观察热损伤后DNA双链断裂形成;台盼蓝拒染实验研究热损伤后细胞存活率;蛋白免疫印记实验检测ATM磷酸化及DNA结合RAD18情况。结果流式结果显示H1299细胞经45℃热损伤1 h后S期细胞较未加热组明显增多(P<0.01);热损伤后Ed U阳性率随时间变化趋势同S期比例变化;S期细胞热损伤后需经37℃正常孵育一定时间方可观察到"彗星拖尾"现象,且随着正常孵育时间延长,反映DNA双链断裂的尾力矩值越大;台盼蓝拒染实验显示S期细胞受热后7.5 h内死亡率保持较低水平,之后细胞死亡加速;热损伤后ATM磷酸化先增多后减少,热损伤导致DNA结合RAD18明显减少。结论细胞热损伤后可发生S期阻滞,阻滞期间因复制继续、DNA损伤修复抑制而延迟形成的致死性DNA双链断裂是S期细胞热敏感的可能原因。
Objective To study the pattern of DNA double-strand break （DSB） formation in S-phase cells after thermal damage and explore the mechanisms behind heat sensitivity of S-phase cells and delayed DSBs. Methods Flow cytometry was used to analyze the cell cycle arrest in H1299 cells exposed to thermal damage, and EdU incorporation assay was employed to evaluate the DNA replication capacity of the cells. The cells synchronized in S phase were obtained by serum starvation and DSBs were observed dynamically using neutral comet assay. Trypan blue dye exclusion technique was used to analyze the cell viability after thermal damage. Western blotting （WB） was used to detect the phosphorylation of ATM and DNA binding RAD18. Results The percentage of S-phase cells increased significantly after exposure of the cells to 45 ℃ for 1 h （P〈0.01）. The time-dependent variation pattern of EdU incorporation was similar to that of S-phase cell fraction. The comet tail began to appear only after incubation of the cells at 37℃for some time and the Olive tail moment （OTM） increased with prolonged incubation. Cell death remained low until 7.5 h after heat exposure of the S-phase cells and then increased rapidly. The phosphorylation of ATM first increased but then decreased drastically. In cells with heat exposure, DNA binding RAD18 was attenuated obviously compared that in non-exposed cells. Conclusion Thermal damage causes cell cycle arrest in S phase, and delayed fatal DSBs occur in the arrested cells due to persistent replication and DNA damage repair suppression, which are the possible cause of heat sensitivity of S-phase cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2016年第4期472-476,共5页
Journal of Southern Medical University
基金
国家自然科学基金(81272407)
卫生部重大科技专项课题资助项目(W2009BX015)~~
