南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (4): 554-560.doi: 10.12122/j.issn.1673-4254.2022.04.11

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过表达TrxR1重组HEK293细胞株的构建和鉴定

吕晓梅，周知音，朱 丽，周 吉，黄慧丹，张 超，刘晓平   

  1. 皖南医学院药物筛选与评价研究所，2附属弋矶山医院生殖医学中心，安徽 芜湖 241000
  • 出版日期:2022-04-20 发布日期:2022-04-29

Construction and identification of a HEK293 cell line with stable TrxR1 overexpression

LÜ Xiaomei, ZHOU Zhiyin, ZHU Li, ZHOU Ji, HUANG Huidan, ZHANG Chao, LIU Xiaoping   

  1. Center of Drug Screening and Evaluation, Wannan Medical College, Wuhu 241000, China; Center for Reproductive Medicine, First Affiliated Hospital of Wannan Medical College, Wuhu 241000, China
  • Online:2022-04-20 Published:2022-04-29

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摘要： 目的 构建稳定过表达硫氧还蛋白还原酶1（TrxR1）的HEK293细胞株，为TrxR1的功能研究以及靶向TrxR1药物筛选提供细胞模型。方法 通过PCR扩增，连接转化以及Sanger双脱氧测序构建并筛选出TrxR1的重组慢病毒表达载体pLVX-PuroTXNRD1，转染HEK293细胞，经嘌呤霉素筛选获得稳定转染细胞株。后续研究分为3组进行：①TrxR1过表达HEK293细胞：pLVX-Puro-TXNRD1载体稳定转染细胞；②对照HEK293细胞：pLVX-Puro空载病毒载体稳定转染细胞；③正常HEK293细胞；通过RT-qPCR、Western blot实验检测上述3组细胞中TrxR1的mRNA以及蛋白表达情况；通过胰岛素终点法以及TRFS-green探针成像检测上述3种细胞内TrxR1的酶活力；通过CCK8实验检测上述3种细胞对TrxR1特异性抑制剂auranofin的敏感性。结果 构建载体经DNA测序，成功获得插入TrxR1的重组慢病毒表达载体pLVX-Puro-TXNRD1。与HEK293以及HEK293-NC细胞相比，HEK293-TrxR1-OE细胞中TrxR1的mRNA和蛋白高表达，且酶活力也同样显著上升（P<0.005）；而与HEK293以及HEK293-NC细胞相比，auranofin对HEK293-TrxR1-OE细胞中TrxR1酶活力以及细胞增殖的抑制效率均显著下降（P<0.005）。结论 通过构建pLVX-Puro-TXNRD1慢病毒载体，成功获得过表达TrxR1酶的HEK293细胞株，该细胞对特异性靶向TrxR1的抑制剂的抗增殖作用具有抵抗，因而可用于靶向TrxR1药物的筛选。

关键词: 硫氧还蛋白还原酶1；慢病毒包装；过表达；细胞模型

Abstract: Objective To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs. Methods TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay. Results TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P<0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P<0.005). Conclusion We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.

Key words: thioredoxin reductase 1; lentiviral packaging; overexpression; cell model