慢病毒载体介导人非小细胞肺癌A549细胞Akt2基因靶向抑制  

Target-specific inhibition of Akt2 in non-small cell lung cancer A549 cell line mediated by lentivirus vector

作　　者：刘丹[1] 黄燚[2] 陈勃江[1] 蒲蓉[3] 曾静[1] 王蕾[4] 李为民[1] 

机构地区：[1]四川大学华西医院呼吸内科,四川成都610041 [2]四川省医学院科学院四川省人民医院检验科,四川成都610041 [3]成都市第三人民医院呼吸内科,四川成都610041 [4]四川大学华西医院中西医结合科,四川成都610041

出　　处：《中华肺部疾病杂志（电子版）》2011年第6期10-14,共5页Chinese Journal of Lung Diseases(Electronic Edition)

摘　　要：目的通过构建慢病毒shRNA表达载体,靶向抑制Akt2基因的表达,获得稳定干扰Akt2基因的细胞克隆,为进一步探讨Akt2的功能奠定基础。方法设计并合成针对Akt2靶基因的双链寡核苷酸序列,经退火、酶切、连接反应,将干扰序列插入pGCsil-GFP质粒,经感受态细胞转化、阳性克隆测序鉴定所插入的片段,经转染及慢病毒包装,收集浓缩病毒进行滴度测定,感染至人肺腺癌细胞A549,通过无菌流式细胞分选GFP强阳性细胞,并应用荧光定量PCR及Western-blot验证干扰效果。结果重组pGCsil-shAkt2-GFP质粒载体经阳性克隆测序鉴定显示插入序列与shAkt2干扰序列完全符合,测定重组慢病毒vshAkt2滴度为3E+9TU/ml。重组慢病毒shAkt2感染A549细胞,经流式细胞术分选90.1%GFP为强阳性细胞群。通过qRT-PCR与western blot检测Akt2mRNA及蛋白表达水平,提示与正常对照组比较,shAkt2组细胞中Akt2mRNA水平下降63.39%,蛋白水平减少91.56%。结论本研究所构建的慢病毒shAkt2表达载体,能够在A549细胞内稳定、有效的抑制靶基因Akt2的表达,为深入研究Akt2在非小细胞肺癌(NSCLC)中的作用奠定了基础。Objective To construct the lentivirus shRNA vector targeting Akt2. Cell clones inhibiting Akt2 expression were obtained, and it offered information for the discussion of Akt2. Method Sequences for targeting the Akt2 gene was selected. The double strand shRNA oli-go was ligated to pGCsil-GFP lentivirus vector. The construct was verified by sequencing. Then a lentiviral expression vector, pHelperl. 0, and p-Helper 2.0 plasmid vectors were cotrasnfected into 293T cells by Lipofectamin 2000. The viral particles were collected and infected A549 cells. After selection of GFP positive cells by FACS, mRNA and protein expression levels of Akt2 were determined by qPCR and western blot, respectively. Results The sequencing results confirmed that pGCsil-shAkt2-GFP vector was successfully constructed. After virus packaging, the concentrated virus titer was 3E ＋ 9 TU/ml. shAkt2 stable transfected cells were purified by FACS with GFP marker. The ratio of selection is 90.1%. Efficacy of Akt2 knockdown was determined by qRT-PCR and western blot. Our data showed that Akt2 mRNA level of shAkt2 group was significantly decreased by 63.39% compared with control groups, and protein expression level of Akt2 was inhibited by 91.56%. Stable knockdown of Akt2 in A549 cells was successfully established. Conclusions shAkt2 can significantly inhibit the Akt2 expression levels in A549 cell. It establi-shes the foundation for the study on effects of Akt2 on NSCLC.

关 键 词：非小细胞肺癌 AKT2 RNA干扰 慢病毒载体 

分 类 号：R734.2[医药卫生—肿瘤]