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建立HBV基因组以附加体形式复制的稳定细胞系用于抗病毒药物评估。

Establishment of stable cell lines in which the HBV genome replicates episomally for evaluation of antivirals.

作者信息

Sun Suofeng, Li Yuan, Liu Bowei, Zhang Bingyong, Han Shuangyin, Li Xiuling

机构信息

Department of Gastroenterology, Henan Provincial People's Hospital, Zhengzhou, China.

Department of Traditional Chinese Medicine, The Third Affiliated Hospital Affiliated of Henan University of Traditional Chinese Medicine, Zhengzhou, China.

出版信息

Arch Med Sci. 2018 Nov 20;16(2):407-413. doi: 10.5114/aoms.2018.79712. eCollection 2020.

Abstract

INTRODUCTION

Due to the increasing resistance to nucleot(s)ide analogs in patients with chronic hepatitis B, development of new antiviral drugs to eradicate hepatitis B virus is still urgently needed.

MATERIAL AND METHODS

To date, most studies on evaluating anti-HBV drugs have been performed using cell lines where the HBV genomic DNA is chromosomally integrated, e.g. Hep2.2.15 in HBV-infected livers of the viral episomal genome replicates in the nucleus and covalently closed circular DNA (cccDNA) serves as a transcriptional template. Another option involves the use of HBV-infected cells of HepaRG or NTCP-overexpressing cells. However, the development of the infection system is expensive and laborious, and its HBV expression level remained low.

RESULTS

Compared to HuH7 cells, the established stable cell lines based on episomal-type pEB-Multi vectors can been expressed HBV wild-type by qRT-PCR and immunoblotting ( < 0.05). These two vectors are also sensitive to Entecavir and against nucleoside analog Lamivudine in mutants cellines.

CONCLUSIONS

It is worth demonstrating how useful the established cell system is for evaluating antiviral agents and their mechanisms of action.

摘要

引言

由于慢性乙型肝炎患者对核苷酸类似物的耐药性不断增加,仍然迫切需要开发新的抗病毒药物来根除乙型肝炎病毒。

材料与方法

迄今为止,大多数评估抗乙肝病毒药物的研究都是使用细胞系进行的,在这些细胞系中,乙肝病毒基因组DNA是染色体整合的,例如在乙肝病毒感染的肝脏中的Hep2.2.15,病毒游离基因组在细胞核中复制,共价闭合环状DNA(cccDNA)作为转录模板。另一种选择是使用HepaRG感染乙肝病毒的细胞或过表达NTCP的细胞。然而,感染系统的开发昂贵且费力,其乙肝病毒表达水平仍然较低。

结果

与HuH7细胞相比,基于游离型pEB-Multi载体建立的稳定细胞系通过qRT-PCR和免疫印迹法能表达乙肝病毒野生型(P<0.05)。这两种载体对恩替卡韦也敏感,并且在突变细胞系中对核苷类似物拉米夫定有抗性。

结论

值得证明所建立的细胞系统在评估抗病毒药物及其作用机制方面有多有用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3293/7069427/d55636f94d20/AMS-16-2-34155-g001.jpg

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