中文摘要：

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。

英文摘要：

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 MTI？ 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 ceils and suppresses the cell growth.