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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。
Objective To investigate the inhibitory effects of different binding arms length 1023 DNAzymes on the expression of hepatitis B virus S gene and C gene in 2.2.15 cells. Methods 1023 DNAzymes with different binding arms length were designed and synthesized, which were directed against the open reading frames A157UG and A1816UG of hepatitis B virus S gene and C gene respectively. Different 1023 DNAzymes were transfected with 2.2.15 cells respectively. The levels of HBsAg and HBeAg in 2.2.15 cell culture supernatant were determined by radioimmunoassay. HBVDNA level in 2.2.15 cell culture supernatant was determined by real-time fluorescence quantitative PCR. MTT assay cytotoxicity. Results 1023DNAzymes with different binding arms length could effectively inhibit the expression of HBsAg and HBeAg in the concentration range of 0.1 ~ 2.5μmol / L. The inhibitory effect could be up to 72h. Under the same dose of the same transfection time, the inhibition rates of 1023 DNAzymes with different binding arms on HBsAg and HBeAg expression showed the following relationship: DrzBS9> DrzBS8> DrzBS7; DrzBC9> DrzBC8> DrzBC7. DrzBS9 and DrzBC9 at 2.5μmol / L dose conditions, 48h after transfection of HBsAg and HBeAg expression inhibition rates were as high as 95% and 92%. 1023DNAzymes with different binding arms had no significant effect on the level of HBVDNA in 2.2.15 cell culture supernatant. MTT cytotoxicity assay showed no toxic effect of 1023 DNAzymes on 2.2.15 cells in the concentration range of 0.1 ~ 2.5μmol / L. Conclusions Different DNA binding lengths of 1023 DNAzymes in 2.2.15 cells inhibited the expression of S gene and C gene of Hepatitis B virus. The inhibitory effects of DrzBS9 and DrzBC9 were stronger.