电离辐射诱导的两种新线粒体DNA缺失的分析鉴定

Detection and confirmation of two novel mitochondrial DNA deletions in human iymphoblastoid cell line irradiated by ^60 Co gamma rays

目的筛选电离辐射诱导的线粒体DNA（mtDNA）缺失类型，并进行鉴定。方法用10Gy^60Coγ射线照射正常人淋巴细胞细胞系，用2对引物的长PCR扩增照射前后样品的mtDNA基因组。发现可能缺失片段后用限制条件的普通PCR进行确认，并对PCR产物进行纯化、克隆、测序和核酸同源性（BLAsT）分析。结果照射前的淋巴细胞系mtDNA样品只扩增出预期全长片段，照射后每对引物还扩增出可能为缺失造成的短片段；进一步限制条件的普通PCR证实了mtDNA缺失的存在。PCR产物经纯化、克隆后测序进行BLAST分析表明两种mtDNA缺失分别为7455bp（重链nt475～7929）、9225bp（重链nt7714～369）的mtDNA缺失，均发生在8bp正向重复序列之间。进一步的生物信息学检索得出两种mtDNA缺失为新发现的mtDNA缺失类型。结论电离辐射诱导出人淋巴细胞mtDNA 7455bp和9225bp缺失。

Objective To screen the radiation induced mitochondtial DNA （mtDNA） deletion and confirm it. Methods Long-range PCR with two pairs of primers, which could amplify the whole human mitochondrial genome, was used to analyze the lymphoblstoid cell line before and after exposed to 10 Gy ^60 Co γ- rays. The limited condition PCR was used to certify the possible mtDNA deletion shown by long-range PCR. The PCR products were purified, cloned, sequenced and the sequence results were BLASTed. Results The predicted bands of mtDNA were observed on the control cell lines, and the possible mtDNA deletions were also detected on the irradiated cell lines. The deletions were certified by the limited condition PCR. The sequence BLAST results of the cloned PCR products showed that there were two kinds of deletions, 7455 bp deletion （nt475-7929 in heavy strand） and 9225 bp deletion （nt7714-369 in heavy strand）, which were both results from 8 bp direct repeats. Further bioinformatics analysis showed that the two deletions were novel deletions. Conclusions Ionizing radiation could induce the 7455 bp and 9225 bp deletions in human lymphocytes.