中国癌症杂志 ›› 2015, Vol. 25 ›› Issue (8): 566-571.doi: 10.3969/j.issn.1007-3969.2015.08.002

• 论著 • 上一篇    下一篇

维甲酸诱导基因G慢病毒表达载体的构建及对肺癌细胞A549的影响

吴军录,权文强,姚懿雯,万海英,李 冬   

  1. 同济大学附属同济医院检验科,上海 200065
  • 出版日期:2015-08-30 发布日期:2015-12-14
  • 通信作者: 李冬 E-mail:186ld@163.com
  • 基金资助:
    国家自然科学基金(81272603,81472179);上海市浦江人才计划(13PJ1407300);2013年教育部留学回国人员科研启动基金;上海申康医院发展中心课题(SHDC22014008)。

Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line

WU Junlu, QUAN Wenqiang, YAO Yiwen, WAN Haiying, LI Dong   

  1. Department of Clinical Laboratory, Tongji Hosptial, Tongji University, Shanghai 200065, China
  • Published:2015-08-30 Online:2015-12-14
  • Contact: LI Dong E-mail: 186ld@163.com

摘要: 背景与目的:维甲酸诱导基因G(retinoic acid-induced gene G,RIG-G)是从急性早幼粒细胞性白血病细胞系NB4细胞中克隆出的肿瘤抑制基因。我们通过调控基因(Tet-on)系统构建受强力霉素(doxycycline,DOX)诱导RIG-G基因表达的A549细胞系,并观察其对A549细胞增殖的作用。方法:采用实时定量PCR(quantitative real-time PCR,qRT-PCR)技术扩增RIG-G基因片段,利用LR重组系统构建pLenti6/TO/V5-GIM-RIG-G慢病毒载体,对该慢病毒载体和Tet-on慢病毒载体包装和病毒滴度测定后,感染A549细胞;采用有限稀释法筛选稳定株;使用细胞免疫荧光和蛋白[质]印迹法(Western blot)鉴定RIG-G基因受DOX调控表达的效果;CCK-8试验检测细胞增殖能力。结果:成功构建pLenti6/TO/V5-GIM-RIG-G慢病毒载体,包装后测得其活性滴度为1.0×108 TU/mL;慢病毒经Tet-on包装后物理滴度为4×109 VP/mL;RIG-G蛋白成功地在慢病毒感染后的A549稳定株中合成和表达,并且受DOX的诱导调控。RIG-G蛋白表达成功后,A549细胞的增殖与对照组相比显著降低(1.168±0.107 vs 2.099±0.162,P<0.05)。结论:本研究成功建立了RIG-G基因可调控表达的A549稳定株;RIG-G蛋白对A549的增殖有抑制作用。

关键词: 维甲酸诱导基因G, 慢病毒, 表达调控, 细胞增殖

Abstract: Background and purpose: Retinoic acid-induced gene G (RIG-G) is a tumor suppressor gene which is cloned by NB4 cell line from a acute promyelocytic leukemia cell. This study aimed to investigate the effect of RIG-G in lung cancer cells A549 by constructing a lentiviral vector expressing RIG-G under doxycycline (DOX) regulation. Methods: RIG-G gene amplification was performed by quantitative real-time PCR (qRT-PCR). pLenti6/ TO/V5-GIM-RIG-G lentiviral vector with GFP was built by LR recombination system. The concentration of pLenti6/ TO/V5-GIM-RIG-G lentiviral vector and Tet-on lentiviral vector were measured by virus titer method. After infecting A549 cells, stably transfected lines were selected via limiting dilution analysis. RIG-G gene expression was examined by immunofluorescence staining and Western blot assay. Cellular proliferation was determined by CCK-8 assay. Results: The concentrations of pLenti6/TO/V5-GIM-RIG-G lentiviral vector and Tet-on lentiviral vector were 1.0×108 TU/mL and 4×109 VP/mL, respectively. RIG-G was expressed in lentivirus infected A549 cells after adding DOX, and the amount of cells with GFP could be observed by fluorescence microscopy. After the expression of RIG-G protein, the proliferation activity of A594 cell was significantly inhibited compared to the control group (1.168±0.107 vs 2.099±0.162, P<0.05). Conclusion: The regulated expression of RIG-G gene was established in A549 lung cancer cell line. The RIGG protein has potential abilities to inhibit the proliferation of lung cancer cell A549.

Key words: Retinoic acid-induced gene G, Lentiviral, Expression regulation, Cell proliferation