10-23 DNAzyme对乙型肝炎病毒X基因表达的抑制作用

Inhibition of 10-23 DNAzyme against hepatitis B virus X gene expression

目的以增强绿色荧光蛋白(EGFP)作为报告分子,探讨10-23 DNAzyme对乙型肝炎病毒(HBV)X基因表达的抑制作用。方法设计并合成3种能针对HBV X基因的特异性10-23 DNAzymes,分别命名为DrzHBVX-7、DrzHBVX-8和DrzHBVX-9,能分别特异地识别HBV X基因开放阅读框(ORF)的A1376UG。将内有HBV X全基因片段(不含终止密码)的融合表达质粒pHBx-EGFP与10-23 DNAzyme共转染AD293细胞作为共转染组,用pHBx-EGFP单独转染AD293细胞作为对照组,分别用荧光显微镜观察并用流式细胞仪检测细胞荧光强度,同时用半定量一步法RT-PCR分析融合荧光蛋白HBx-EGFP mRNA的相对表达量。结果各共转染组细胞荧光蛋白HBx-EGFP的表达均明显弱于对照组(P<0．05);各试验组HBx-EGFP mRNA的相对表达量与对照组比较也均明显减少(P<0．05);各共转染组细胞之间HBx-EGFP的表达及HBx-EGFP mRNA的相对表达量差异无统计学意义(P>0．05)。结论10-23 DNAzyme对HBV X基因的表达有特异抑制作用,在HBV感染的基因治疗中会有潜在的应用价值。

Objective To explore the inhibition effects of 10-23 DNAzyme on hepatitis B virus （HBV） X gene expression. Methods 10-23 DNAzymes named DrzHBVX-7, DrzHBVX-8 and DrzHBVX-9 specific to HBV X gene ORF A^1376 UG were designed and synthesized. With enhanced green fluorescent protein （EGFP） as report molecule, pHBx-EGFP containing HBV X-EGFP fusion gene and 10-23 DNAzymes were cotransfected into AD293 cells as cotransfection groups, and pHBx-EGFP alone transfected into AD293 cells as control group, respectively. Inhibition effects of HBV X gene expression by 10-23 DNAzymes were observed accordingly. Expression of EGFP was examined by fluorescent microscopy and flow cytometry analysis. EGFP mRNA levels were analyzed by semi-quantitative RT-PCR （reverse-transcription polymerase chain reaction）. Results expressions of HBx-EGFP in cotransfection groups were less than those in control group observed by fluorescent microscopy and detected by flow cytometry （P 〈 0.05）. The expressions of HBx-EGFP mRNA in cotransfection groups were less than those in control group detected by semi-quantitative RT-PCR （P 〈 0.05）. The expression of HBx-EGFP and HBx-EGFP mRNA in each cotransfection group had no significant difference （P 〉 0.05）. Conclusion 10-23 DNAzyme possessed the specific inhibition effects of HBV X gene expression. This would play a potent antiviral role in HBV gene therapy.