【摘要】 目的构建针对人Cox-2基因的小干扰RNA(siRNA)表达载体,观察RNA干扰对肺癌细胞Cox-2基因表达的沉默作用。方法设计Cox-2靶向的发夹状siRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入pSINsi-hU6载体,转化扩增后进行序列测定。用脂质体包裹转染人肺癌细胞NCI-H446,采用RT-PCR检测Cox-2基因mRNA表达的变化。结果把针对Cox-2基因的siRNA的双链寡核苷酸片段克隆入pSINsi-hU6载体,经过酶切鉴定与测序,结果正确;RT-PCR检测显示,Cox-2基因的表达水平明显降低。结论成功构建出显著沉默细胞Cox-2基因表达的siRNA载体。更多还原

【Abstract】 Objective To construct a small interfering RNA(siRNA) expression vector targeting human Cox-2 gene and to observe the effects of gene silencing of Cox-2 by RNA interference on lung carcinoma cells. Methods Hairpin-shaped small interference RNA targeting Cox-2 gene was designed and the complementary single strands of DNA fragments were synthesized and annealed.The DNA fragments were recombinated into pSINsi-hU6 vector,followed by amplification and DNA sequencing,then transfected into human lung carcinoma cell line NCI-H446 mediated by Liposome.The change of the Cox-2 gene mRNA expression was detected by RT-PCR. Results The DNA fragments encoding Cox-2 targeted siRNA were cloned into the pSINsi-hU6 and confirmed by restrictive enzyme digestion and DNA sequencing.RT-PCR revealed a strongly decreased expression level of Cox-2. Conclusion A siRNA vector which can silence Cox-2 gene expression effectively has been successfully constructed.更多还原