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

发布时间：2018-05-05 13:11

本文选题：阿德福韦酯 + Hep　；参考：《解放军医学杂志》2017年09期

【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。
[Abstract]:Objective to observe the occurrence of classical drug resistance mutation in Hep G2.2.15 cells induced by different concentrations of adefovir dipivoxil, and to explore the mechanism of drug resistance. Methods Hep G2.2.15 cells were cultured in 12-well plate with or without 0.1 渭 mol/L ADV of 0.1 渭 mol/L ADV. The cells were subcultured every 3 days and cultured to 110th generation. The HBV DNA of cells and supernatants were extracted and sampled once every 10 generations. The reverse transcriptase region of HBV ccc DNA in cell and HBV DNA in supernatant was amplified by ATP dependent DNA enzyme digestion and rolling loop amplification method and single tube nested PCR method, respectively. Direct sequencing of PCR products and cloning sequencing (20 clones / samples) were used to analyze whether the RT region of HBV ccc DNA and supernatant HBV DNA produced classical ADV resistant mutations. Results HBV DNA.0.01 渭 mol/L ADV group and 0.1 渭 mol/L ADV group continued to express HBV DNA.0.01 渭 mol/L ADV and 0.1 渭 mol/L ADV with the prolongation of ADV intervention time. The intracellular and supernatant HBV DNA load of total HBV DNA CCC DNA and supernatant decreased continuously .0.01 渭 mol/L ADV group and the medium to 110th generation of supernatant did not detect the resistance mutation in 0.1 渭 mol/L ADV group and supernatant. RT region 181V N236T mutation. 1.0 渭 mol/L ADV was detected in RT region at 110th generation. The cells in the group were inhibited from culture at the 15 th generation. Conclusion there is a HBV ccc DNA cycle cell in Hep G2.2.15 cells. The classical drug-resistant mutation of Hep G2.2.15 cells can be induced by continuous culture medium containing appropriate concentration of ADV.
【作者单位】：桂林医学院研究生院;解放军302医院临床研究管理中心;
【基金】：国家自然科学基金面上项目(81371852)~~
【分类号】：R96

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