反基因及反义寡核苷酸抑制乙型肝炎病毒复制与表达的研究

Inhibition of hepatitis B virus replication and synthesis of antigen by antigene and antisense oligodeoxynucleotides in vitro

目的 探讨反基因及反义寡核苷酸抗乙型肝炎病毒(HBV) 作用。方法 设计合成针对HBV 核心启动子Sp1 位点(1734 nt～1754 nt)及前CRNA、前基因组RNA5′端起始区(1814 nt～1834 nt) 的21 聚硫代修饰反基因寡核苷酸(ODNan21)及反义寡核苷酸(ODNas21) 。将各寡核苷酸与22 .1 .5 细胞温育。采用酶联免疫吸附法(ELISA) 及斑点杂交法分别检测经处理的22.1 .5 细胞培养上清HBsAg、HBeAg 及HBV DNA 水平。结果 ODNan21 、ODNas21 处理的22 .1 .5 细胞HBsAg、HBeAg 水平明显低于对照组( P< 0 .001)。浓度为10 μmol/L 时,ODNan21,ODNas21 对HBsAg、HBeAg 的抑制分别达57.% 、77 % ;61% 、79 .6 % ;两者联合给药的抑制达71 .5% 、85 % 。该抑制呈剂量依赖性并于给药后48 小时达高峰。ODNan21 、ODNas21 处理的22 .1.5 细胞HBVDNA 水平低于对照组。无关序列对照寡核苷酸对HBVDNA 及抗原合成无明显影响。在实验范围内,寡核苷酸对22 .1 .5 细胞无毒性作用。结论 ODNan21、ODNas21 在体外能有效抑制HBV 复制及抗原合成。

Objective To study the antiviral effect of antigene oligodeoxynucleotides (ODNan) and antisense oligodeoxynucleotides (ODNas) on replication of hepatitis B virus(HBV). Methods A 21mer phosphorothioate ODNan (ODNan21) directed at Sp1 sites in hepatitis B virus(HBV) core promoter (1734 nt～1754 nt) and a 21mer phosphorothioate ODNas (ODNas21) complementary to the initiation site of pre C mRNA and pregenomic RNA (1813 nt～1833 nt) were synthesized. 22.1.5 cells were treated with these oligodeoxynucleotides. HBsAg, HBeAg, and HBV DNA were determined by enzyme linked immunosorbent assay(ELISA) and dot hybridization methods, respectively, in the treated 22.1.5 cells. Results The levels of HBsAg and HBeAg in ODNan21 group and ODNas21 group were lower than that in blank control group ( P <0.001). ODNan21 and ODNas21 of 10 μmol/L reduced synthesis of HBsAg, HBeAg by 57.5% and 61%, 77% and 79.6%, respectively, while the combination of ODNan21 and ODNas21 showed stronger inhibition to HBsAg and HBeAg (71.5% and 85%). The inhibitory effects were dose dependent, and reached to the highest point after 48 hours of administration. The HBV DNA was significantly decreased in the ODNan21 group as well as ODNas21 group. No inhibitory effects on the levels of HBsAg, HBeAg and HBV DNA were observed in the oligodeoxynucleotide control group. In virus inhibitory concentrations the oligodeoxynucleotides were harmless to 22.1.5 cells. Conclusion The results indicated that both antigene oligodeoxynucleotides and antisense oligodeoxynucleotides could partly inhibit the replication of HBV and synthesis of antigen.