解放军医学杂志2017，Vol.42Issue(9)：753-758,6.DOI:10.11855/j.issn.0577-7402.2017.09.01
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
Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir
摘要
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.关键词
阿德福韦酯/HepG2.2.15细胞/共价闭合环状DNA/耐药变异Key words
adefovirdipivoxil/HepG2.2.15 cells/covalently closed circular DNA/drug-resistant mutation分类
医药卫生引用本文复制引用
李奇,杨媛,李乐,蔡少平,苗福珍,李韦杰,刘妍,徐东平..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[J].解放军医学杂志,2017,42(9):753-758,6.基金项目
国家自然科学基金面上项目(81371852)This work was supported by the National Natural Science Foundation of China (81371852) （81371852）
