摘要
目的探讨DNA依赖蛋白激酶亚基(DNA-PKcs)基因沉默后对肝癌耐药细胞Bel-7402/5-Fu DNA损伤修复功能的影响。方法采用阳离子脂质体法将sh DNA-PKcs干扰质粒转染Bel-7402/5-Fu细胞,根据荧光细胞数计算转染率,实时荧光定量聚合酶链反应(q RT-PCR)及Western blot检测DNA-PKcs的沉默效率;Western blot检测TopoⅡ、γ-H2AX蛋白表达;5-乙炔基-2'脱氧尿嘧啶核苷法检测细胞DNA合成。结果质粒的转染效率为67%;q RT-PCR及Western blot检测结果显示,DNA-PKcs m RNA及蛋白水平的沉默效率分别为82.6%和71.8%;Western blot结果显示,实验组TopoⅡ蛋白表达水平比对照组低,差异有统计学意义(P<0.05),γ-H2AX蛋白表达水平比对照组高,差异有统计学意义(P<0.01)。5-乙炔基-2'脱氧尿嘧啶核苷法结果显示,实验组DNA合成率比对照组低,差异有统计学意义(P<0.05)。结论 sh DNA-PKcs干扰质粒能下调Bel-7402/5-Fu细胞中DNA-PKcs、TopoⅡ蛋白的表达,抑制Bel-7402/5-Fu细胞DNA合成。
Objective To investigate the effect of shDNA-PKcs on DNA damage repair function of the muhidrug-resistant hepatocellular carcinoma cell line Be17402/5-Fu. Methods shDNA-PKcs plasmids were transiently transfected into Be17402/5-Fu cells via cathodolyte Jiposome transfection method. Transfection effi- ciency was evaluated by calculating the fluorescent cells and silence efficiency was determined using qRT- PCR and Western blot. qRT-PCR and Western blot were used to detect mRNA and protein expressions of Topo Ⅱ and y-H2AX. The cellular DNA synthesis was detected by 5-ethynyl-2＇-deoxyuridine （EdU）. Results Transfection efficiency was 67%. qRT-PCR and Western blot results showed the efficiency of RNA and pro- tein interference for DNA-PKcs was 82.6% and 71.8%. Western blot analysis showed that the protein expres- sion of Topo 11 in the shDNA-PKcs group was significantly lower than that in the control group （P 〈 0.05）, while y-H2AX expression was higher than that of the control group （P〈 0.01）. EdU detection results showed the DNA synthesis of the shDNA-PKcs group decreased compared with that of the control group （P 〈 0.05）. Conclusions shDNA PKcs interference plasmid can down-regulate the expressions of DNA-PKcs and Topo Ⅱ and inhibit the DNA synthesis of Be17402/5-Fu cells.
出处
《中国现代医学杂志》
CAS
北大核心
2016年第17期19-22,共4页
China Journal of Modern Medicine
基金
贵州省社发攻关项目(No:[2009]3066)
贵州省社发攻关项目[黔科合SY(2013)3008]
