针对HBVX基因的反义核酸抑制HBV表达的体外研究

An in vitro study on inhibition of hepatitis B virus replication and expression by antisense oligodeoxynucleotides directed at HBV X translation initiator

目的筛选反义脱氧寡核苷酸 (ASON)抗乙型肝炎病毒 (HBV)的有效靶向位点。方法设计合成针对HBVX翻译起始位点的ASON即序列Ⅰ ,并设非互补序列作对照即序列Ⅱ ,以人肝癌细胞系 (2 .2 .15 )为细胞模型 ,应用ELISA动态观察不同浓度 (1、5、10 μmol/L)ASON一次性用药对HBV基因表达的抑制作用 ,用MTT比色法观察ASON对细胞代谢的影响 ,以苔盼蓝染色计数观察ASON对细胞活性的影响。结果ASON可有效地抑制HBV基因的表达 ,在 1、5、10 μmol/L浓度时对HBsAg的抑制率分别为 6 3%、45 %和 5 7% ,对HBeAg的抑制率分别为 38%、41%和 5 6 % ;而对照序列Ⅱ对HBsAg和HBeAg的抑制率均低于 12 %。MTT法表明ASON对 2 .2 .15细胞代谢无影响(P >0 .0 5 ) ,苔盼蓝染色计数表明ASON对 2 .2 .15细胞活性无影响 (P >0 .0 5 )。

PurposeThe aim is to select out effective target region of antisense oligonucleotides inhibiting HBV gene expression.MethodsWe designed and synthesized ASON directed at HBV X translation initiator. Noncomplementary sequence was taken as control. 2.2.15 cells in human hepatoma cell line were chosen as a model of culture in vitro . We have consecutively observed that ASON (1?5?10 μmol / L) inhibits the effect on HBV gene expression by ELISA. The effect of ASON on 2.2.15 cells metabolism was detected by MTT assay. The effect of ASON on 2.2.15 cell viability was microscopically done after trypan blue staining.ResultsThe result showed that ASON at 1?5?10 μmol / L concentration was able to effectively inhibit HBV gene expressing HBsAg and HBeAg. Their inhibitory rates of HBsAg were 63% ,45% and 57%, respectively. Their inhibitory rates of HBeAg were 38%,41% and 56%, respectively. Noncomplementary sequence could not effectively inhibit HBV gene expressing HBsAg and HBeAg. Their inhibitory rates of HBsAg and HBeAg were low by 12%. MTT results showed that ASON had no effect on 2.2.15 metabolism ( P >0.05). viable cells count after trypan blue staining showed that ASON had no effect on 2.2.15 viability( P >0.05).ConclusionHBV X translation initiator is an effective target region of ASON inhibiting HBV gene expression.