反基因锁核酸体外抑制乙肝病毒前S1基因表达

 目的 探讨针对乙肝病毒前S1基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用。方法 针对乙肝病毒前S1基因同聚嘌呤区，利用RNAstructure软件分别设计合成锁核酸、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转染HepG2.2.15细胞，采用荧光定量聚合酶链反应技术（FQ-PCR）、时间分辨免疫荧光技术（TRFIA）和酶联免疫法（ELISA）分别监测1、3、5和7 d细胞培养上清液中HBV DNA、HBsAg和前S1抗原的含量;四甲基偶氮唑蓝（MTT）法检测锁核酸对细胞代谢的影响。结果 加入锁核酸后，对HBV DNA复制、HBsAg和前S1抗原表达均显示有较强的抑制作用，且抑制率随时间呈增高趋势，7 d后抑制率分别达64.32%、67.51%和63.88%。LNA对细胞代谢无明显影响。结论 针对乙肝病毒前S1基因同聚嘌呤区的反基因锁核酸，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。

Hepatitis B virus(HBV) preS1 gene-specific anti-gene locked nucleic acid(LNA) significantly inhibits preS1 gene expression in vitro

Objective To investigate the inhibitory effects of hepatitis B virus(HBV) preS1 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 HBV preS1 gene were designed, synthesized, and transfected by cationic liposomes into HepG2 2.2.15 cells. The HBsAg,preS1-Ag and HBV DNA of supernatant was tested by time-resolved fluorescence immune assay(TRFIA) ,real-time fluorescent quantitative PCR(FQ-PCR) and enzyme linked immunosorbent assays(ELISA) at 1, 3, 5 and 7 d after treatment. LNA’s cyto-toxicity on cell was evaluated by MTT method. Results The anti-gene LNA that targeting on the purine rich region of HBV preS1 gene showed strong inhibitory effects on replication of HBV DNA and the expression of HBsAg and preS1-Ag with the inhibition rates of 65.99%,67.49% and 63.88% respectively after 7 days.There’s no obvious toxicity on cell. Conclusion anti-gene locked nucleic acid(LNA) that targeting on the purine rich region of HBV preS1 gene has show strong inhibition on HBV in vitro.It has a therapeutic potential in the treatment of patients infected with HBV.