反基因锁核酸体外阻断肝癌细胞系乙肝病毒S基因表达

Anti-gene locked nucleic acid( LNA) inhibits HBV S gene expression in vitro

目的探讨针对乙肝病毒S基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的效果。方法针对乙肝病毒S基因同聚嘌呤区设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体介导,体外转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术和时间分辨免疫荧光技术分别监测2、4、6、8和10 d细胞培养上清液中HBV DNA、HBsAg和HBeAg的含量;四甲基偶氮唑蓝法检测锁核酸对细胞代谢的影响。结果加入锁核酸后,对细胞内HBV DNA复制、HBsAg与HBeAg表达均有较明显的时间和剂量依赖性抑制作用,6 d后抑制率分别为52.14%、57.48%和29.63%。锁核酸对细胞代谢无明显影响。结论针对乙肝病毒S基因同聚嘌呤区的锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据。

Objective To investigate the inhibitory effects of hepatitis B virus （HBV） S gene-specific anti-gene locked nucleic acid（LNA） on HBV replication and expression in hepG2.2. 15 cells. Methods The anti-gene LNA which were complementary to the purine rich region of the HBV S gene was synthesized and transfected into HepG2 2.2. 15 cells by cationic liposomes. The HBsAg, HBeAg and HBV DNA of supernatants were tested by time-re- solved fluorescence immune Assay （TRFIA） and real-time fluorescent quantitative polymerase chain reaction （FQ- PCR） at 2, 4, 6, 8 and 10 d after treatment. LNA＇s cyto-toxicity on cell was evaluated by methyl thiazolyl tetrazo- lium （MTT） method. Results The anti-gene LNA targeting at the purine rich region of HBV S gene showed signifi- cantly inhibitory effects on replication of HBV DNA and the expression of HBsAg and HBeAg with the inhibition rates of 52. 14%, 57. 48% and 29. 63% respectively after 6 days. There＇s no obvious toxicity on cell. Conclusions Anti-gene locked nucleic acid targeting at the purine rich region of HBV S gene has shown significant inhibition on HBV in vitro. It has a therapeutic potential for the treatment of patients infected with HBV.