| 过表达TrxR1重组HEK293细胞株的构建和鉴定 |
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| 引用本文： | 吕晓梅,周知音,朱 丽,周 吉,黄慧丹,张 超,刘晓平.过表达TrxR1重组HEK293细胞株的构建和鉴定[J].南方医科大学学报,2022,42(4):554-560. |
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| 作者姓名： | 吕晓梅 周知音 朱 丽 周 吉 黄慧丹 张 超 刘晓平 |
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| 作者单位： | 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 |
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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。
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| 关 键 词： | 硫氧还蛋白还原酶1 慢病毒包装 过表达 细胞模型 |
Construction and identification of a HEK293 cell line with stable TrxR1 overexpression |
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| Lü Xiaomei,ZHOU Zhiyin,ZHU Li,ZHOU Ji,HUANG Huidan,ZHANG Chao,LIU Xiaoping.Construction and identification of a HEK293 cell line with stable TrxR1 overexpression[J].Journal of Southern Medical University,2022,42(4):554-560. |
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| Authors: | LÜ Xiaomei ZHOU Zhiyin ZHU Li ZHOU Ji HUANG Huidan ZHANG Chao LIU Xiaoping |
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| Affiliation: | 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 |
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| 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. |
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| Keywords: | thioredoxin reductase 1 lentiviral packaging overexpression cell model |
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