摘要
目的利用RNA干扰（RNAi）技术将针对性构建的重组质粒转染人人肺癌细胞株NCI-H520，观察下调Pokemon基因对人肺癌细胞生物学行为的影响。方法利用Ambion公司设计软件设计针对Pokemon基因的2条小干扰RNA（siRNA）序列，并合成2对互补的siRNA编码DNA片段，定向克隆至RNAi-ReadypSIREN-RetroQ载体的BamHI和EcoRI位点，构建重组体质粒，分别命名为pSIREN-Pokemon-1和pSIREN-Pokemon-2，同时设立转染空载体对照组。Westernblot检测人肺癌细胞中Pokemon蛋白表达，噻唑蓝（M1vr）比色实验检测转染后细胞的增殖抑制结果，流式细胞分析技术检测转染后细胞周期的分布。结果利用RNAi-Ready pSIRENR RetroQ载体构建的重组质粒可以成功抑制人肺癌细胞NCI-H520中Pokemon蛋白的表达。Westernblot检测发现人肺癌细胞NCI-H520中Pokemon蛋白高水平表达，转染后其表达水平明显下降。转染后第3天，M1Tr比色实验显示pSIREN-Pokemon-1组（0．0015±0．0030）和pSIREN-Pokemon-2组（0．0086±0．0015）与pSIREN-Pokemon.0组（0．0230±0．0010）比较差异有统计学意义（P〈0．05），而pSIREN-Pokemon-1与pSIREN-Pokemon-2组之间差异无统计学意义（P〉0．05）。流式细胞分析检测发现转染siRNA细胞与转染空载体细胞的细胞周期差异无统计学意义（P〉0．05）。pSIREN-Pokemon-1转染组及pSIREN-Pokemon-2转染组形成克隆的数量（18．0±0．3、16．0±0．4）明显低于pSIREN-Pokemon-O组（52．0±0．4，P〈0．05）。结论使用pSIREN载体成功构建的重组质粒转染人肺癌细胞可有效抑制原癌基因Pokemon的表达。转染后细胞增殖受到抑制，生长速度减慢。抑制Pokemon蛋白表达对细胞周期无明显影响。靶向siRNA可以存人肺癌细胞中抑制Pokemon蛋白表达，负调节肺癌细胞生长。
Objective The aim of this research was to observe the inffluence of the low expression of Pokemom gene to biological behaviour of human lung cancer cell after transfeeting the targeted recombi nant plasmid into human lung cancer cell line NCI-H520 by using RNA interference （RNAi） technology. Methods Two strips targeted small interfering RNA （siRNA） sequences, which is aimed at the Pokemon gene, were designed by using the design software of Ambion company, and two pairs of complementary DNA fragments of siRNA were also synthesized, then cloned into BamH I and EcoR I of the RNAi-Ready pSIREN-RetroQ vector, which were named as pSIREN-Pokemon-1 and pSIREN-Pokemon-2, respectively. The control group was set at the mean time. Western blotting was used to test the expression of Pokemon protein, Methyl thiazol tetrazolium （MTT） was to the inhibition of cell proliferation, and flow eytometry was for the cell cycle analysis. Results （ 1 ） The recombinant plasmids which were constructed by RNAi-Ready pSIREN-RetroQ vector suecessfully inhibited the expression of Pokemon protein in human lung eaucer cell NCI-H520. The detective results of Western blotting showed the high expression level of Pokemon protein in NCI-H520, but the expression level decreased obviously after transfection. （2） The results of MTr revealed that there was significantly proliferation in pSIREN-Pokemon-1 （0. 001 5 ± 0. 003 0）and pSIREN-Pokemon-2 （0. 008 6±0. 001 5） in comparison with pSIREN-Pokemon-0 （0. 023 0 ±0. 001 0, P 〈0. 05）. 3 days after transfection, whereas there was no significant difference between pSIREN-Pokemon-1 与 pSIREN-Pokemon-2 （P 〉 0. 05）. （3） Flow eytometry analysis showed that there was no significant difference between transfeet- ed cells and control group which were transfected blank vector into cells. （4） Cell clone forming experi- ments suggested that the number and size of clone both reduced compared with control group. Conclusion （ 1 ） The recombinant plasmids which were co
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第4期791-793,共3页
Chinese Journal of Experimental Surgery
