Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir
10.11855/j.issn.0577-7402.2017.09.01
- VernacularTitle:阿德福韦酯持续干预对HepG2.2.15细胞经典耐药突变的诱导作用
- Author:
Qi LI
1
,
2
;
Yuan YANG
;
Le LI
;
Shao-Ping CAI
;
Fu-Zhen MIAO
;
Wei-Jie LI
;
Yan LIU
;
Dong-Ping XU
Author Information
1. 541004 广西桂林 桂林医学院研究生院
2. 100039 北京 解放军302医院临床研究管理中心
- Keywords:
adefovirdipivoxil;
HepG2.2.15 cells;
covalently closed circular DNA;
drug-resistant mutation
- From:
Medical Journal of Chinese People's Liberation Army
2017;42(9):753-758
- CountryChina
- Language:Chinese
-
Abstract:
Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.
