DNA-PKcs反义核酸增强HeLa细胞对辐射及化疗药物顺铂的敏感性

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，Western blot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-PKcs 5’端320bp cDNA片段，重组于tet启动子控制表达质粒pCl^neo-Tet-splice，转染Hela细胞，获得3个稳定转化克隆；Western blot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。

Increased sensitivity of HeLa cells to UV radiation and chemotherapy drug cisplatin by antisense RNA of DNA-PKcs

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.