重离子辐射引起的线粒体DNA突变定量分析

Quantitative Analysis of Mitochondrial DNA Mutations Induced by Heavy Ion Radiation

目的对重离子辐射引起的线粒体DNA突变进行定量分析。方法使用X射线和重离子束对人乳腺癌细胞MCF-7进行辐照,对照后细胞进行克隆存活分析;用real-time PCR定量检测线粒体DNA4 977缺失,克隆测序法定量检测线粒体D310区突变。结果克隆存活结果和辐照后D310多态性分布显示重离子射线对MCF-7细胞的增殖抑制效果比X射线更为明显,引起的D310突变也更多,并且这些突变体能在辐照后的MCF-7细胞中稳定积累,但重离子辐照引起的线粒体DNA 4 977 bp缺失在细胞中仅能短暂存在。结论辐照后的细胞中存在着克隆选择机制,严重影响线粒体的正常功能突变体短暂存在于辐照后的MCF-7细胞,但很快被清除,如4 977大片段缺失;而D310突变随着细胞遗传,这表明它在线粒体中没有重要功能。

Objective To quantitative analyze heavy-ion induced mitochondrial DNA mutations at different doses of irradiation. Methods Clonogenic survival of human breast cancer cells MCF- 7 was measured after X-rays or carbon ions radiation. Mitochondrial DNA 4977 deletions were detected by real-time PCR and D310 point mutations were detected by cloning sequencing, respectively. Results Clonogenic survival results and D310 polymorphism distribution after radiation showed that carbon ions radiation inhibited MCF-7 cells proliferation more effectively and caused more D310 mutations than X-ray radiation, moreover, these mutants were stably accumulated in MCF-7 clones after radiation, while mtDNA 4977 bp deletions induced by carbon ions radiation only temporally exist in irradiated MCF-7 cells. Conclusion A clonal-selection mechanism exists in cells after radiation. Certain mutants that affect the normal functions of mitochondria seriously could temporally existed in irradiated MCF-7 cells, then will be eliminated soon, such as 4977 deletions; whereas D310 mutants are inherited, which is indicative of its irrelevance to the mitochondrial function.