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,q RT-PCR)技术扩增RIG-G基因片段,利用LR重组系统构建p Lenti6/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p Lenti6/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/m L;慢病毒经Tet-on包装后物理滴度为4×109 VP/m L;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。
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 ofRIG-G in lung cancer cells A549 by constructing a lentiviral vector expressing RIG-G under doxycycline （DOX） regulation.Methods：RIG-G gene ampliifcation was performed by quantitative real-time PCR （qRT-PCR）. pLenti6/TO/V5-GIM-RIG-G lentiviral vector withGFP was built by LR recombination system. The concentration of pLenti6/TO/V5-GIM-RIG-G lentiviral vector andTet-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 immunolfuorescence 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 andTet-on lentiviral vector were 1.0×10^8TU/mL and 4×10^9 VP/mL, respectively. RIG-G was expressed in lentivirus infected A549 cells after adding DOX, and the amount of cells withGFP could be observed by lfuorescence microscopy.After the expression of RIG-G protein, the prolif-eration activity of A594 cell was signiifcantly inhibited compared to the control group （1.168±0.107vs 2.099±0.162, P〈0.05）.Conclusion：The regulated expression ofRIG-G gene was established in A549 lung cancer cell line. The RIG-G protein has potential abilities to inhibit the proliferation of lung cancer cell A549.
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2015年第8期566-571,共6页
China Oncology
基金
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)
关键词
维甲酸诱导基因G
慢病毒
表达调控
细胞增殖
Retinoic acid-induced gene G
Lentiviral
Expression regulation
Cell proliferation
