利用基因缺陷细胞模型探讨hOGG1和hMTH1的替补作用  

A study of the substitution effect of hOGG1 and hMTH1 in oxidative DNA damage with gene-deficient cell strains

作　　者：柯跃斌[1] 吴双[1] 黄娟[1] 袁建辉[1] 邓平建[1] 程锦泉[1] 

机构地区：[1]深圳市疾病预防控制中心现代医学分子生物学重点实验室,518055

出　　处：《中华预防医学杂志》2014年第3期197-202,共6页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（81072323/H2607,30872149/C1504）;深圳市科委基础研究计划项目（JC201105170713A）

摘　　要：目的 通过建立基因缺陷细胞模型,探讨修复基因hOGG1与hMTH1在DNA氧化性损伤中的作用与关系.方法 利用慢病毒感染人胚肺成纤维细胞（HFL）,分别建立hOGG1基因和hMTH1基因缺陷细胞模型.将HFL细胞在100 μmol/L的H2O2中孵育12h,分别利用高压液相色谱串联电化学检测技术及RT-PCR技术分析8-羟基-脱氧鸟嘌呤（7,8-dihydro-8-oxoguanine,8-oxo-dG）水平及hOGG1和hMTH1基因表达水平.结果 用高滴度慢病毒感染靶细胞后得到hOGG1基因和hMTH1基因缺陷细胞模型,hOGG1 mRNA表达水平（0.09±0.02）比正常HFL细胞（1.00±0.04）下降了91％,hMTH1 mRNA表达水平（0.41±0.04）比正常HFL细胞（1.02±0.06）下降了60％；用100 μmol/L的H2O2诱导12 h,hOGG1基因缺陷细胞的hMTH1基因表达水平（1.26±0.18）相比对照组（1.01±0.07）提高了25％,hMTH1基因缺陷细胞的hOGG1基因表达水平（1.54±0.25）提高了52％；hOGG1基因缺陷细胞的8-oxo-dG水平（2.48±0.54）是对照组（0.80±0.16）的3.1倍,差异有统计学意义（P＜0.01）,hMTH1基因缺陷细胞（1.84±0.46）的8-oxo-dG水平是对照组（0.80±0.16）的2.3倍,差异均有统计学意义（P＜0.01）.结论 利用基因缺陷细胞模型,发现在氧化诱导作用下,修复基因hOGG1与hMTH1在DNA损伤修复活动中可能分别表现出一定的协同性替补作用,hOGG1的替补作用强于hMTH1.Objective To investigate the potential substitution effect of hOGG1 and hMTH1 on oxidative DNA damage,based on gene-deficient cell strains models.Methods hOGG1 and hMTH1 gene deficient cell strains models were established by Human embryonic lung fibroblasts（HFL） cells.After HFL cells being exposed to 100 μmol/L H2O2 for 12 h,HPLC-EC detecting technique and RT-PCR method were adopted to analyze the genetic expression level of 8-oxo-dG （7,8-dihydro-8-oxoguanine）.Results The gene-deficient cell strains models of hOGG1 and hMTH1 were obtained by infecting target cells with high titer of lentivirus.The mRNA expression level of hOGG1 was 0.09 ±0.02,91% lower than it in normal HFL cells,which was 1.00 ± 0.04.As the same,the mRNA expression level of hMTH1 （0.41 ± 0.04） also decreased by 60% compared with it in normal HFL cells （1.02 ± 0.06）.After induced by 100 μmol/L H2O2 for 12 h,the genetic expression level of hMTH1 in hOGG1 gene-deficient cells （1.26 ± 0.18） increased 25% compared with it in control group （1.01 ± 0.07）.Meanwhile,the genetic expression level of hOGG1 in hMTH1 gene-deficient cells （1.54 ± 0.25） also increased by 52%.The DNA 8-oxo-dG levels in hOGG1 gene-deficient cells （2.48 ± 0.54） was 3.1 times compared with it in the control group （0.80 ± 0.16）,the difference showed statistical significance （P 〈0.01）.Whereas the 8-oxo-dG levels in hMTH1 gene-deficient cells （1.84 ± 0.46） was 2.3 times of it in the control group,the difference also showed statistical significance （P 〈 0.01）.Conclusion Based on gene-deficient HFL cells models,a synergetic substitution effect on DNA damage and repair activity by both hOGG1 and hMTH1 were firstly discovered when induced by oxidation.The substitution effect of hOGG1 were stronger than that of hMTH1.

关 键 词：RNA干扰 基因缺陷 DNA氧化 修复基因 生物标志 替补作用 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]