【摘要】 目的探讨不同结合臂长1023DNAzymes在2.2.15细胞内对乙型肝炎病毒S基因和C基因表达的抑制作用。方法设计并合成不同结合臂长的1023DNAzymes,能分别针对乙型肝炎病毒S基因和C基因开放阅读框A157UG和A1816UG。不同的1023DNAzymes分别转染2.2.15细胞,放射免疫分析法测定2.2.15细胞培养上清中HBsAg和HBeAg水平,实时荧光定量PCR法测定2.2.15细胞培养上清中HBVDNA水平。MTT法检测细胞毒性。结果不同结合臂长的1023DNAzymes在0.1～2.5μmol/L浓度范围内均能有效抑制HBsAg和HBeAg的表达,抑制效应可长达72h。在同一剂量相同转染时间的条件下,不同结合臂长的1023DNAzymes对HBsAg和HBeAg表达的抑制率呈以下关系:DrzBS9>DrzBS8>DrzBS7;DrzBC9>DrzBC8>DrzBC7。DrzBS9和DrzBC9在2.5μmol/L剂量条件下,转染48h后对HBsAg和HBeAg表达的抑制率可分别高达95%和92%。不同结合臂长的1023DNAzymes对2.2.15细胞培养上清中HBVDNA水平并无明显影响。MTT细胞毒性检测表明,在0.1～2.5μmol/L浓度范围内未见1023DNAzymes对2.2.15细胞的毒性作用。结论不同结合臂长1023DNAzymes在2.2.15细胞内对乙型肝炎病毒S基因和C基因表达均有一定的抑制作用,DrzBS9和DrzBC9的抑制作用较强。更多还原

【Abstract】 Objective To explore the inhibition effects of 10-23 DNAzymes with different substrate-recognition domains targeting hepatitis B virus (HBV) S gene and C gene expression in 2.2.15 cells. Methods 10-23 DNAzymes with different substrate-recognition domains specific to HBV S gene open reading frame (ORF) A 157UG and HBV C gene ORF A 1816UG were designed and synthesized, respectively. Different 10-23 DNAzymes were transfected into 2.2.15 cells which is a stable HBV producing cell line. HBsAg and HBeAg secreted into culture media were detected by radioimmunoassay (RIA) and HBV DNA levels were measured by real-time PCR. 3-(4,5-dimethylthiagol-2-yl)-2,5-drphnyl tetragolium brornide (MTT) assays were performed to evaluate cytotoxicity. Results HBsAg and HBeAg expressions were reduced by various DNAzymes (0.1-2.5 μmol/L) with different substrate-recognition domains after transfection. The antiviral effects of DNAzymes were apparent until 72 h post-transfection. The inhibition rates of the DNAzymes at the same dose on HBsAg and HBeAg in the same period of post-transfection were as the following: DrzBS-9>DrzBS-8>DrzBS-7; DrzBC-9>DrzBC-8>DrzBC-7. Among all the DNAzymes used, DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent, with HBsAg and HBeAg reduced 95% and 92% 48 h post-transfection at the dose of 2.5 μmol/L, respectively. The inhibition effects on HBV DNA by various DNAzymes with different substrate-recognition domains were of no significance. There were no evident cytotoxic effects of these DNAzymes in the range from 0.1 to 2.5 μmol/L. Conclusion 10-23 DNAzymes with different substrate-recognition domains targeting HBV S gene and C gene mRNA possessed specific inhibition effects in 2.2.15 cells, and DrzBS-9 targeting S gene and DrzBC-9 targeting C gene were most potent.更多还原