论文部分内容阅读
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,具有较大应用潜力
Objective To investigate the anti-hepatitis B virus (HBV) effect of anti-sense and antisense oligonucleotides. Methods 21 polythio-modified antisense oligodeoxynucleotides (SNPs) were designed and synthesized based on the HBV core promoter Sp1 (1734 nt ~ 1754 nt) and pre-CRNA and the 5 ’end of the pregenomic RNA (1814 nt ~ 1834 nt) ODNan21) and antisense oligonucleotide (ODNas21). Each oligonucleotide was incubated with 22.1.5 cells. The levels of HBsAg, HBeAg and HBV DNA in the cultured supernatant of 22.1. 5 cells were detected by enzyme-linked immunosorbent assay (ELISA) and dot blot hybridization. Results The levels of HBsAg and HBeAg in 22. 1. 5 cells treated with ODNan21 and ODNas21 were significantly lower than those in the control group (P <0 .001). The inhibitory rates of ODNan21 and ODNas21 to HBsAg and HBeAg were 57.%, 77%, 61% and 79. 6% respectively at the concentration of 10 μmol / L. The combination of ODNan21 and ODNas21 reached 71.5% and 85%, respectively. The inhibition was dose-dependent and reached its peak 48 hours after dosing. ODNan21, ODNas21 treated 22.1.5 cells HBVDNA levels lower than the control group. The irrelevant sequence control oligonucleotides had no significant effect on HBVDNA and antigen synthesis. Within the experimental range, oligonucleotides have no toxic effect on 22.1.5 cells. Conclusion ODNan21 and ODNas21 can effectively inhibit HBV replication and antigen synthesis in vitro, which has great potential for application