【摘要】 目的构建N-Ras基因小发卡结构RNA(shRNA)干扰真核表达质粒载体,并初步观察其对二羟环氧苯并芘(BPDE)转化的人支气管上皮细胞(16HBE-T)生长的影响。方法根据GenBank提供的N-Ras cDNA序列,设计并合成shRNA寡核苷酸片段,与含U6启动子的pGPU6/GFP/Neo质粒定向连接,构建4个真核表达载体,并经限制性内切酶酶切和DNA测序进行鉴定。转染16HBE-T细胞48 h后,采用逆转录-聚合酶链反应(RT-PCR)和Western blot检测重组质粒对N-Ras基因表达的影响;用噻唑蓝法(MTT)观察重组质粒对细胞生长的抑制作用。结果构建了4个N-Ras shRNA真核表达载体,经限制性内切酶酶切和DNA测序证实与设计完全一致。构建的4个表达载体pGPU6/GFP/Neo-NRas-566、pGPU6/GFP/Neo-NRas-305、pGPU6/GFP/Neo-NRas-613和pGPU6/GFP/Neo-NRas-791分别转染16HBE-T细胞48 h后,RT-PCR显示N-Ras基因mRNA表达水平依次下调95%、70%、92%和60%;Western blot显示蛋白表达依次降低92%、45%、58%和34%。MTT发现16HBE-T细胞的生长受到明显抑制,且与N-Ras基因表达水平正相关。结论成功构建了N-Ras基因特异性shRNA真核细胞表达载体,有效沉默了N-Ras在16HBE-T细胞中的表达,抑制了恶性转化细胞的生长。更多还原

【Abstract】 Objective To construct eukaryotic expression vector of small hairpin RNA(shRNA) of N-Ras and investigate the effect of recombinant plasmid on suppressing growth of malignantly transformed human bronchial epithelial cell induced by anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide(BPDE)(16HBE-T).Methods Four shRNAs of N-Ras gene were designed and synthesized,and inserted into plasmid pGPU6/GFP/Neo including U6 promoter.The recombinant expression vectors were identified by restriction map and sequence analysis,and transfected into 16HBE-T cells.After 48h of transfection,RT-PCR and Western blot were conducted to assess the efficency of N-Ras silencing;and MTT was used to observe the proliferation of 16HBE-T cells.Results The four recombinant plasmids were verified by restriction map and sequence analysis.The sequences completely coincided with the designs.After 48h of transfection with pGPU6/GFP/Neo-NRas-566,pGPU6/GFP/Neo-NRas-305,pGPU6/GFP/Neo-NRas-613 and pGPU6/GFP/Neo-NRas-791 recombinant plasmid,RT-PCR showed that the N-Ras mRNA expression was decreased by 95%,70%,92% and 60%,respectively,and Western blot exhibited that its protein expression was decreased by 92%,45%,58% and 34%,correspondingly.MTT demonstrated the growth of 16HBE-T transfected cells was suppressed significantly,and its proliferation ability correlated with N-Ras expression level.Conclusion\The shRNA eukaryotic expression vector against N-Ras gene is successfully constructed.It effectively silences the expression of N-Ras in 16HBE-T cells and suppresses the cell growth.更多还原