MTH1基因的低表达对HeLa细胞内RNA氧化程度的影响  

Oxidization Level Analysis of RNA in MTH1 Knocking Down HeLa Cell.

作　　者：戴大鹏[1] 干伟[1] 张立群[1] 聂犇[2] 徐仁爱[2] 蔡剑平[1] 

机构地区：[1]卫生部北京医院/卫生部北京老年医学研究所卫生部老年医学重点实验室,100730 [2]温州医学院药学院药理教研室,325035

出　　处：《医学研究杂志》2011年第6期35-38,共4页Journal of Medical Research

基　　金：科技部国际科技合作计划项目(2006DFB31410);卫生部北京医院面上项目(BJ-2008-77)

摘　　要：目的建立敲减MTH1基因的HeLa细胞稳定细胞株,研究MTH1基因的低表达对HeLa细胞内RNA氧化程度的影响。方法设计并合成针对MTH1基因的3条siRNA,分别转染HeLa细胞,选择干扰效果最为理想的靶序列连接入反转录病毒载体Retro-Q,在293T细胞内包装病毒颗粒,将病毒转染HeLa细胞后使用嘌呤霉素筛选抗性克隆株,用western b lot技术检测克隆株的MTH1的表达量以确定干扰效果最理想的稳定细胞株,用API5000型质谱仪检测稳定细胞株RNA中的8-oxoG与G的含量以评价RNA的氧化程度。结果本研究设计的3条siRNA中有2条干扰效率可达90%以上,所建立的敲减MTH1基因HeLa细胞稳定细胞株的干扰效果达80%以上,质谱检测结果表明MTH1基因低表达的稳定细胞株每106个G中含有14.9个8-oxoG,而对照细胞中则仅含9.7个8-oxoG。结论 MTH1基因的低表达可引起HeLa细胞中RNA氧化程度明显升高。Objective To construct cell lines with decreased levels of MTH1 expression, and to investigate the influence of lowering MTH1 expression on the oxidation level of RNA in HeLa cells. Methods Three siRNA targeting to MTH10RF region was designed and synthesized. Target sequences with the highest RNA interface efficiency were then introduced into retrovirus vector Retro - Q and used for the package of virus particles which could infect HeLa cells to get the resistant cell clones. Western blot analysis was carried out to screen most reasonable stable cell lines with highest RNAi efficiency. APIS00 mass spectrometer was used for the detection of 8 - oxoG and G amounts at last in order to evaluate the oxidation level of RNAo Results Two of three siRNA could get more than 90% knocking down effi-ciency and at last we got several stable cell lines with knocking down efficiency more than 80%. We found that RNA of MTH1 knocking down stable cell lines contained 14.9 8 -oxoG per 106 G, whereas control cells contained 9.7 oxoG per 106 G. Conclusion Decreased MTH1 expression in HeLa cell could increase the oxidation level of RNA.

关 键 词：8-oxoG RNA干扰 MTH1 

分 类 号：R737.33[医药卫生—肿瘤]