摘要
背景:线粒体DNA4977bp缺失的累积对衰老的多细胞动物来说是一个显著的特征,同时也与各种肿瘤细胞的转移和凋亡有关。目前的研究已证明辐射可特异性地诱导此缺失的产生,有望将其作为新的DNA水平的辐射损伤生物剂量标记。目的:用聚合酶链反应方法检测碳离子辐射引起的Hela细胞线粒体DNA4977bp突变缺失,并观察碳离子辐射剂量及辐射时间与线粒体DNA4977bp缺失的相关性。设计、时间及地点:细胞DNA水平的对照实验,于2008-10/11在甘肃省兰州市近代物理研究所完成。材料:人宫颈癌细胞Hela细胞株。方法:Hela细胞经2～8Gy的碳离子辐射后,于2～24h各个时间点分别提取包含线粒体DNA的全基因组DNA,并用聚合酶链反应法扩增线粒体DNA4977bp缺失的特异片段,通过限制反应模板浓度和聚合酶链反应循环数的方法进行定性分析。主要观察指标:碳离子辐射剂量及辐射时间对线粒体DNA4977bp缺失的影响。结果:经2Gy辐照处理的Hela细胞在2,4,8,24h4个时间点的检测中均无聚合酶链反应阳性产物,8Gy辐照处理的Hela细胞在12,16,24h3个时间点可检测到线粒体DNA4977bp缺失,且随时间延长而累积。辐照后12hHela细胞在8Gy辐照处理后可检测到线粒体DNA4977bp缺失,辐照后16和24hHela细胞在6,8Gy辐照处理后可检测到线粒体DNA4977bp缺失。结论:碳离子辐射引起的Hela细胞线粒体DNA4977bp缺失存在剂量相关性,且其累计程度可能与辐射时间有关。
BACKGROUND： Mitochondrial DNA （mtDNA） deletion at 4 977 bp is a noticeable feature in senile multicellular animals, and is related to various tumor cell metastasis and apoptosis. Studies have demonstrated that irradiation could specifically induce deletion, and may become a novel radiation injury biomarker at DNA level.
OBJECTIVE： To detect carbon ion irradiation-induced common mtDNA deletion by PCR and investigate the correlation of irradiation dosage and duration to mtDNA deletion at 4 977 bp. DESIGN, TIME AND SETTING： Control cell experiment was performed at Institute of Modem Physics in Lanzhou from October to November 2008. MATERIALS： Human cervical carcinoma cell strain （Hela）.
METHODS： Hela cells were irradiated with 2-8 Gy ^12C^6＋ ions respectively, and collected at 2-24 hours after irradiation. Whole genome DNA including mtDNA was extracted at each time point and the deletion segment was amplified using PCR amplification for template. The DNA template concentration was equaled in each reaction and PCR cycles were restricted to 28 cycles to get to a semi-quantity result.
MAIN OUTCOME MEASURES： Influence of irradiation dosage and duration to mtDNA deletion at 4 977 bp.
RESULTS： Hela cells at the four time points （2, 4, 8, 24 hours） after 2 Gy irradiation cannot detect positive PCR products, mtDNA 4 977 bp deletion was detected in Hela cells at 12, 16, and 24 hours after 8 Gy irradiation, and the accumulation presents does dependent manner, mtDNA 4 977bp deletion was detected in Hela cells at 12, 16, 24 hours after 8 Gy irradiation, and 16, 24 hours after 6Gy irradiation.
CONCLUSION： The common mtDNA deletion in Hela cells induced by carbon ion irradiation exhibits does dependent pattern, and the accumulation of the common deletion related to irradiation duration.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第9期1665-1668,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金重点项目(10835011)
国家自然科学基金面上项目(10675151)
甘肃省2007年重大科技专项项目(O702NKDA045)
甘肃省2008年重大科技专项项目(0801NKDA001)
兰州市科技攻关项目(07-2-07)~~
