慢病毒介导的shRNA靶向干扰nestin鼻咽癌稳定细胞株的建立

目的检测nestin基因在8种鼻咽癌细胞中的表达差异,寻找高表达的细胞株。构建nestin基因的shRNA表达载体,建立nestin稳定干扰的鼻咽癌细胞株。方法应用Real-time PCR和免疫荧光技术检测nestin在鼻咽癌细胞株中的表达水平。构建带GFP的重组靶向nestin shRNA慢病毒表达质粒,筛选阳性克隆,测序鉴定。293T细胞包装病毒,收集病毒上清,测定滴度。经Real-time PCR内源筛靶,选择最佳靶点。将重组慢病毒感染5-8F细胞,Western blotting和Real-time PCR检测nestin的表达。结果在8种鼻咽癌细胞株中,5-8F细胞中nestin表达较高。PCR和测序验证重组质粒构建成功。慢病毒感染5-8F细胞后,与阴性对照组和空白组相比,nestin的mRNA和蛋白水平明显受到抑制。结论成功构建了干扰nestin的慢病毒重组质粒,并建立了稳定靶向干扰nestin表达的5-8F鼻咽癌细胞株。

Establishment of a nasopharyngeal carcinoma cell line with stable nestin silencing by lentivirus-mediated RNA interference

Objective To establish a nasopharyngeal carcinoma(NPC) cell line with stable nestin gene silencing induced by short-hairpin RNA(shRNA) interference.Methods The nestin mRNA levels in 8 NPC cell lines were detected by real-time PCR and immunofluorescence assay.The recombinant lentiviral shRNA expression plasmid targeting nestin was packaged into mature lentivirus by 293T cells,and the supernatant containing the virus was harvested,concentrated and titrated.The best target for RNA interference was selected by ...