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目的 研究人肿瘤细胞DNA LigaseⅣ基因突变与放射敏感性的关系。方法 常规集落形成方法测定人鼻咽鳞癌 (CNE)、肺腺癌 (SPC A1)和乳腺腺癌 (MCF 7)细胞系不同剂量照射后的细胞存活分数 ,采用聚合酶链式反应和克隆技术测定 3种细胞参与DNA损伤修复的DNAligaseⅣ基因序列 ,分析它们的同源性变化和突变对基因产物亲疏水性的影响。结果 3种细胞存活参数比较存在较大差异 ,CNE细胞较SPC A1和MCF 7细胞显示出较高的放射敏感性。CNE、SPC A1和MCF 7细胞LigaseⅣ基因同源性分别为 99.95 %、99.99%和 99.98% ,包括碱基转换和颠换突变。部分突变碱基导致氨基酸替代并影响基因产物的亲疏水结构。CNE细胞LigaseⅣp的 313aaHis→Arg、5 38aaGly→Arg、5 79aaLys→Arg和 5 85aaAsn→Ser替代与CNE细胞放射敏感性高于SPC A1和MCF 7细胞有关。结论 LigaseⅣp在非同源性末端连接 (NHEJ)途径中起关键和最终连接作用 ,该基因突变影响肿瘤细胞的双链断裂损伤修复能力和放射敏感性。
Objective To study the relationship between DNA LigaseⅣ gene mutation and radiosensitivity in human tumor cells. Methods The routine colony formation assay was used to determine the cell viability of human nasopharyngeal squamous cell carcinoma (CNE), lung adenocarcinoma (SPC A1) and breast adenocarcinoma (MCF 7) cells after irradiation at different doses. Polymerase chain reaction and cloning techniques The DNAligaseⅣgene sequences involved in DNA damage repair were determined and their homology changes and the effects of the mutations on the hydropathicity of gene products were analyzed. Results There were significant differences in survival parameters between the three kinds of cells. CNE cells showed higher radiosensitivity than SPC A1 and MCF7 cells. The homologies of Ligase Ⅳ gene in CNE, SPC A1 and MCF 7 cells were 99.95%, 99.99% and 99.98% respectively, including base conversion and transversion. Partial mutation of the base results in amino acid substitutions and affects the hydrophilic and hydrophobic structure of the gene product. 313aaHis → Arg, 538aaGly → Arg, 579aaLys → Arg and 585aaAsn → Ser of LigaseⅣp in CNE cells were associated with higher radiosensitivity of CNE cells than SPC A1 and MCF 7 cells. Conclusion LigaseⅣp plays a key and final role in the non-homologous end-joining (NHEJ) pathway, which affects the repair ability and radiosensitivity of double-stranded DNA.