摘要
目的构建表达DNA-PKcs反义核酸的真核表达载体,建立DNA-PKcs表达被反义核酸抑制的细胞模型。方法采用RT-PCR扩增DNA-PKcs的cDNA片段,DNA重组技术构建四环素控制表达的真核表达载体;利用Lipofectamine介导基因转染,Westernblot分析鉴定反义核酸抑制DNA-PKcs表达的细胞模型;细胞克隆形成法和细胞生长曲线分析细胞对UV和顺铂的敏感性。结果克隆了DNA-PKcs5'端320bpcDNA片段,重组于tet启动子控制表达质粒pCIneo-Tet-splice,转染Hela细胞,获得3个稳定转化克隆;Westernblot结果表明细胞中DNA-PKcs表达受到反义核酸的抑制,并且细胞对紫外线辐射和顺铂敏感性增加。结论成功建立了DNA-PKcs表达受到反义核酸抑制的细胞模型,抑制DNA-PKcs表达提高了细胞的辐射和顺铂的敏感性。
[Objective]To construct the DNA-PKcs antisense RNA expression vector, establish the cell model of antisense RNA-mediated DNA-PKcs suppression. RT-PCR was used to amplify DNA-PKcs cDNA. pCIneo-tet-splice was used as expression vector. Gene transfection was mediated by Lipofectamine. DNA-PKcs expression was detected by Western Blotting, the cellular sensitivity to UV radiation and cisplatin was analyzed by means of colony-forming ability of growth curve. A 320 bp of DNA-PKcs upstream cDNA was cloned from HeLa cells and inserted in reverse orientation into the expression vector pCIneo-tet-splice, in which the expression of inserted gene was regulated by tetracycline. Three stable transfectants were selected, and Western Blotting analysis indicated that the expression of DNA-PKcs was suppressed by the antisense RNA. The DNA-PKcs antisense RNA markedly increased the sensitivity of HeLa cells to UV radiation as well as cisplatin. [Conclusion] The cell model of DNA-PKcs suppressed expression by antisense RNA was established, and suppression of DNA-PKcs increased cellular sensitivity to UV and cisplatin.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2005年第8期1148-1151,共4页
China Journal of Modern Medicine
基金
本课题受国家自然科学基金(30270423)项目
湖南省科技厅基金(03SSY3104)项目资助
