[1]周飞飞,杨南飞,余文,等.Nrf2 过表达与敲减 Hep1-6稳转细胞株的建立[J].江苏农业学报,2016,(06):1251-1255.[doi:doi:10.3969/j.issn.1000-4440.2016.06.009]
　ZHOU Fei-fei,YANG Nan-fei,YU Wen,et al.Establishment of Nrf2 over-expression and knock down Hep1-6 cell lines[J].,2016,(06):1251-1255.[doi:doi:10.3969/j.issn.1000-4440.2016.06.009]
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Nrf2 过表达与敲减 Hep1-6稳转细胞株的建立()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年06期
页码:
1251-1255
栏目:
遗传育种·生理生化
出版日期:
2017-02-07

文章信息/Info

Title:
Establishment of Nrf2 over-expression and knock down Hep1-6 cell lines
作者:
周飞飞; 杨南飞; 余文; 张冬梅; 丰秀静
（南京大学生命科学院医药生物技术国家重点实验室，江苏南京210046)
Author(s):
ZHOU Fei-fei; YANG Nan-fei; YU Wen; ZHANG Dong-mei; FENG Xiu-jing
(State Key Laboratory of Pharmaceutical Biotechnology，School of Life Science，Nanjing University，Nanjing 210046，China)
关键词:
核因子NF-E2相关因子2(Nrf2); Hep1-6细胞; Nrf2过表达稳转株; Nrf2敲减稳转株; Western blotting; 定量PCR
Keywords:
nuclear factor NF-E2 related factor 2 (Nrf2); Hep1-6 cell; Nrf2 over expression cell line; Nrf2 knock down cell line; Western blotting; qPCR
分类号:
Q786
DOI:
doi:10.3969/j.issn.1000-4440.2016.06.009
文献标志码:
A
摘要:
核因子NF-E2相关因子2(Nrf2)是治疗非酒精性脂肪肝(NAFLD)的一个重要靶点分子。为了从体外研究小分子通过靶向Nrf2改善NAFLD的分子机制，首先PCR克隆鼠源Nrf2基因至pLenti6/V5-D-TOPO空载体得到重组载体pLenti6/V5-Nrf2，通过测序、酶切鉴定正确后，扩繁质粒,制备慢病毒并将其感染Hep1-6肝癌细胞，用稻瘟醇胚素筛选、鉴定获得稳定表达Nrf2的细胞株。同时，用Nrf2干扰质粒构建慢病毒感染Hep1-6肝癌细胞，通过嘌呤霉素筛选、鉴定Nrf2敲减的细胞株。Q-PCR检测结果显示，过表达稳转株(pLenti6/V5-Nrf2)中Nrf2基因的表达量为对照组(pLenti6/V5-LacZ)的4倍，敲减稳转株中Nrf2基因的表达量大幅度降低。Western blotting显示过表达稳转株的Nrf2表达量明显高于对照组，敲减稳转株的Nrf2表达量明显低于对照稳转株，此结果与Q-PCR结果一致。所克隆的Nrf2基因和敲减基因在Hep1-6小鼠肝癌细胞中得到了正确转录和翻译，稳转细胞株构建成功。
Abstract:
Nuclear factor NF-E2 related factor 2 (Nrf2) is an important target molecule in nonalcoholic fatty liver disease (NAFLD). To study the role of small molecules that targets Nrf2 in vitro, mice Nrf2 gene cloned by PCR was cloned into pLenti6/V5-D-TOPO empty carrier,building a pLenti6/V5-Nrf2 reorganization plasmid which was identified via sequencing and restriction enzyme digestion. The plasmid was packed into lentivirus and used to infect Hep1-6 liver cancer cells. Meanwhile, the shRNA-Nrf2 plasmid was packed into lentivirus and used to infect Hep1-6 liver cancer cells as well. The cells were screened with blasticidin or puromycin for about 1 month. The qPCR results showed that the expression of Nrf2 was four times than that of control in Nrf2 over-expression cells while it was significantly reduced in Nrf2 knock down cells. Consistently, the level of protein in the two cell lines detected by Western blotting was obviously increased in Nrf2 over-expression cells or reduced in Nrf2 knock down cells. The results suggest that the Nrf2 over expression and knock down Hep1-6 cell lines have been successfully built and it supplies a useful tool to study the role of drug targeting Nrf2 in vitro.

参考文献/References:

[1]DAY C P, JAMES O F W. Steatohepatitis: a tale of two “hits”?[J]. Gastroenterology, 1998, 114(4): 842-845.

[2]HIROTSU Y, KATSUOKA F, FUNAYAMA R, et al. Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks[J]. Nucleic Acids Research, 2012, 40(20): 10228-10239.
[3]CHORLEY B N, CAMPBELL M R, WANG X, et al. Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha[J]. Nucleic Acids Research, 2012,40(15):7416-7429.
[4]AGYEMAN A S, CHAERKADY R, SHAW P G, et al. Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles[J]. Breast Cancer Research and Treatment, 2012, 132(1): 175-187.
[5]MALHOTRA D, PORTALES-CASAMAR E, SINGH A, et al. Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis[J]. Nucleic Acids Research, 2010, 38(17): 5718-5734.
[6]MACLEOD A K, MCMAHON M, PLUMMER S M, et al. Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds[J]. Carcinogenesis, 2009, 30(9): 1571-1580.
[7]PAEK J, LO J Y, NARASIMHAN S D, et al. Mitochondrial SKN-1/Nrf mediates a conserved starvation response[J]. Cell Metabolism, 2012, 16(4): 526-537.
[8]JUNG K A, CHOI B, NAM C W, et al. Identification of aldo-ketoreductases as NRF2-target marker genes in human cells[J]. Toxicology Letters, 2013, 218(1): 39-49.
[9]WU K C, CUI J Y, KLAASSEN C D. Beneficial role of Nrf2 in regulating NADPH generation and consumption[J]. Toxicological Sciences, 2011,123(2):590-600.
[10]VALDECANTOS M P, PRIETO-HONTORIA P L, PARDO V, et al. Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes[J]. Free Radical Biology and Medicine, 2015, 84: 263-278.
[11]BHALA N, IBRAHIM KAMAL JOUNESS R, BUGIANESI E. Epidemiology and natural history of patients with NAFLD[J]. Current Pharmaceutical Design, 2013, 19(29): 5169-5176.

备注/Memo

备注/Memo:
收稿日期：2016-04-21 基金项目：国家自然科学基金项目(81503082) 作者简介：周飞飞（1987-），河南新乡人，硕士，主要从事化合物药效学评价。(Tel)15751865756；（E-mail）15751865756@163.cn 通讯作者：丰秀静，（Tel）025-89682995；（E-mail）fengxj@nju.edu.cn
更新日期/Last Update: 2017-02-07