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

作　　者：李奇 杨媛[2] 李乐 蔡少平[2] 苗福珍[2] 李韦杰 刘妍[2] 徐东平[2] 

机构地区：[1]桂林医学院研究生院,广西桂林541004 [2]解放军302医院临床研究管理中心,北京100039

出　　处：《解放军医学杂志》2017年第9期753-758,共6页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金面上项目(81371852)~~

摘　　要：目的观察Hep G2.2.15细胞在不同浓度阿德福韦酯(ADV)持续诱导下发生经典耐药突变的情况,并探讨其产生耐药的机制。方法用不含或含0.01、0.1、1.0μmol/L ADV的培养基于12孔板中培养Hep G2.2.15细胞,每3d传代,共培养至110代,分别抽提细胞和上清中的HBV DNA,每10代取样1次。以不降解质粒的ATP依赖性DNA酶消化+滚环扩增+跨缺口PCR方法和单管巢式PCR方法分别扩增细胞内HBV ccc DNA和上清中HBV DNA的反转录酶(RT)区。以PCR产物直接测序结合克隆测序法(20个克隆/样本)分析细胞内HBV ccc DNA和上清HBV DNA的RT区是否产生经典ADV耐药突变。结果未经药物处理的对照组持续稳定表达HBV DNA。0.01μmol/L ADV组和0.1μmol/L ADV组随着ADV干预时间的延长,细胞内总HBV DNA、ccc DNA及上清中HBV DNA的载量持续下降。0.01μmol/L ADV组细胞内和上清中至110代仍未检测出耐药变异,0.1μmol/L ADV组细胞内和上清中在110代时检测到RT区发生rt A181V+N236T变异。1.0μmol/L ADV组由于细胞生长受到抑制于第15代时停止培养。结论 Hep G2.2.15细胞内存在HBV ccc DNA循环池,用含适量浓度ADV的培养基持续培养可以诱导Hep G2.2.15细胞发生经典耐药突变。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 levelsp supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 Ixmol/L and 0.1 ~xmol/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 the 110th 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 Hep G2.2.15细胞 共价闭合环状DNA 耐药变异 

分 类 号：R512.62[医药卫生—内科学]