增殖细胞核抗原突变体的稳定高表达对HeLa细胞顺铂敏感性的影响  

Effects of Overexpression of Mutant PCNA on Cisplatin-Induced Cytotoxicity in HeLa Cells

作　　者：位全芳[1] 曾益军[1] 杨劲[1] 周虎传[1] 刘洋[2] 徐志刚[2] 袁方[2] 赵友光[2] 

机构地区：[1]第三军医大学基础部生物化学与分子生物学教研室,重庆市400038 [2]第三军医大学西南医院全军泌尿外科研究所,重庆市400038

出　　处：《医学分子生物学杂志》2008年第6期498-502,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.30770460);全军医学科研"十一五"计划国际合作项目(No.06H026)~~

摘　　要：目的建立稳定高表达增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)或突变体PC-NA(mutant PCNA,mPCNA)目的蛋白的宫颈癌细胞系HeLa细胞,观察PCNA在顺铂诱导的DNA损伤修复中的作用。方法采用显性负突变策略,通过构建重组人PCNA或mPCNA的真核表达质粒pCDNA3.1/V5-His A-PCNA(His-PCNA)和pCDNA3.1/V5-His A-mPCNA(His-mPCNA),稳定转染到HeLa细胞中,G418筛选建立稳定高表达目的蛋白的HeLa细胞系;Western印迹法检测蛋白的表达情况;MTT法测定顺铂损伤后不同细胞系的细胞存活率。结果真核表达质粒经酶切、测序分析与实验设计完全一致;稳定建系后,Western印迹结果显示在相应位置可见清楚的目的条带;MTT结果显示稳定高表达突变体PCNA的细胞系与对照组相比,其细胞存活率呈明显下降趋势。结论成功构建了真核表达质粒His-PCNA和His-mPC-NA;建立了稳定高表达该质粒的HeLa细胞系;稳定高表达突变体PCNA的细胞系对顺铂损伤更敏感。Objective To explore the role of PCNA in cisplatin-induced DNA damage response. Methods With the strategy of dominant negative mutant, the full length cDNA fragment of human PCNA or mutant PCNA （mPCNA） was cloned into the eukaryotic expression vector pCD- NA3.1/V5-His A separately. The plasmid was each transfected into Hela cells. Following G418 resistant selection, the cell line stably expressing PCNA or mutant PCNA was established. The cytotox- icity of cisplatin was detected with MTF assay. Results The eukaryotic expression plasmids were identified by restriction enzyme and sequencing. The interesting protein was detected by western blot. It was shown that the cells stably expressing mPCNA were much more sensitive to cisplatin compared to the wild type cells. Conclusions The eukaryotic expression plasmids were constructed and the stably transfected cells were established. The mutant PCNA overexpressing Hela cells were more sensitive to cisplatin induced cytotoxicity.

关 键 词：增殖细胞核抗原 稳定转染 HELA细胞 顺铂 

分 类 号：Q786[生物学—分子生物学]