【摘要】 目的探讨小干预RNA(siRNA)对稳定表达人乳头瘤病毒(HPV)6bE6基因的细胞株中靶基因的干预作用。方法建立并筛选稳定表达靶基因HPV6bE6的阳性细胞株,分别设计同源于HPV6bE6基因的不同序列的4对siRNA,采用实时定量PCR技术检测siRNA转染前后细胞内靶mRNA的表达情况。结果转染细胞中HPV6bE6基因表达最低有90%以上被抑制,低浓度siRNA水平(1nmol/L~150nmol/L)即能有效抑制靶基因表达,靶mRNA的沉默于转染siRNA后24h达到最强,且至少可维持4d。结论siRNA-HPV6bE6可以高效、特异地沉默HPV6bE6基因,可能为治疗HPV6b型感染提供实验室依据。更多还原

【Abstract】 Objective To investigate the inhibition of pathogenic viral gene expression in HPV6bE6-positive cell line by specific siRNA, which might have great potential for clinical use. Methods B16 cells were transfected with recombinant plasmid pcDNA3.1(+)-GFP/HPV6bE6, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. Four specific siRNAs, none of which shares homology with exons of known human genes, were designed and synthesized to target HPV6bE6 mRNA. Quantitative real-time PCR was performed to measure the inhibition rates of target gene expression by comparing HPV6bE6 mRNA concentrations before siRNA transfection with those after transfection. The inhibition rates of target gene expression with different siRNA concentrations of 0.2 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L, 150 nmol/L and with different treatment time at 24 h, 48 h, 72 h and 96 h after transfection were measured respectively. Results More than 90% reduction of HPV6bE6 mRNA was observed following treatment with HPV6bE6-siRNA, and HPV-negative cells were apparently unaffected by HPV6bE6-siRNA. The decrease of HPV6bE6 mRNA was maximal at 24 h after siRNA treatment and sustained for at least 4 days. The minimal level of siRNA to efficiently silence the homogeneous target gene HPV6bE6 was 1 nmol/L. Conclusion HPV6bE6-siRNA can efficiently and specifically silence target genes and may be developed as a potential therapeutic approach for HPV infection.更多还原