反基因锁核酸体外阻断肝癌细胞系乙肝病毒S基因表达简

 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，也为反基因治疗治疗提供理论和实验依据。

Hepatitis B virus(HBV) S gene-specific anti-gene locked nucleic acid(LNA) significantly inhibits S gene expression in vitro

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 were designed,synthesized,and transfected by cationic liposomes into HepG2 2.2.15 cells. The HBsAg, HBeAg and HBV DNA of supernatants was tested by time-resolved 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 tetrazolium(MTT) method. Results The anti-gene LNA that targeting on the purine rich region of HBV S gene showed strong 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 that targeting on the purine rich region of HBV S gene has show strong inhibition on HBV in vitro.It has a therapeutic potential in the treatment of patients infected with HBV.