摘要
目的探讨稳定表达的siRNA抑制DNA-PKcs基因表达对胰腺癌细胞BxPC-3放射敏感性的影响。方法以人胰腺癌细胞BxPC-3为研究对象,将预先设计的siRNA与质粒连接。构建质粒与阴性对照质粒分别转化大肠杆菌,经克隆、扩增、纯化后转染BxPC-3细胞,潮霉素筛选,以半定量Westernblot检测靶蛋白表达变化,用细胞克隆计数实验检测放射敏感性变化。结果成功构建以DNA-PKcs基因mRNA为靶序列的pSilencer2.1质粒,经潮霉素筛选出稳定的整合目的质粒的BxPC-3细胞,DNA-PKcs蛋白被抑制最大达到83%,实验组D0、Dq及SF2(1.284±0.017、4.006±0.023和0.567±0.045)均明显低于阴性对照组(1.458±0.026、4.840±0.019和0.833±0.076)(P<0.001)。SER为1.47。结论针对DNA-PKcs基因的siRNA表达载体稳定转染人胰腺癌细胞BxPC-3,能够引发DNA-PKcs表达抑制,进而产生放射增敏作用。
Objective To discuss the radiosensitization effect of DNA- PKcs gene silencing in BxPC-3 cell line by RNA interference（RNAi）. Methods One confirmed siRNA targeting mRNA of human DNA-PKcs gene was synthesized and inserted into plasmid pSilencer 2. 1-U6, which, along with control plasmid, was transformed into DH5a, separately. After amplification, the plasmids were purified and sequenced to determine whether the ligation between siRNA insert and the vector was correct before being transfected into BxPC-3 cells. These cells were screened by hygromycin and named pSilencer 2.1-DNA-PKcs and pSilencer 2. 1-C, separately. Protein level of DNA-PKcs was detected by Western blot. Survival curves for each cell line were obtained by measuring the colony-forming abilities of irradiated cell populations. Results SiRNA was successfully ligated into plasmid pSilencer2, 1-U6. Protein level of DNA-PKcs decreased maximally to 83%. D0, Dq and SF2（1. 284 ± 0. 017, 4. 006 ± 0. 023 and 0. 567 ± 0. 045） of pSilencer2.1-DNA-PKcs transfected BxPC-3 cells were significantly lower than those of pSilencer-C（1. 458 ± 0. 026, 4. 840±0.019 and 0.833± 0.076）（P〈0.001）. SER was 1.47. Conclusions SiRNA produced by stable transfection can silence DNA-PKcs gene and radiosensitize BxPC-3 cell.
出处
《胰腺病学》
2006年第2期88-91,共4页
Chinese JOurnal of Pancreatology
