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目的探讨针对甲型病毒(influenza Avirus,IFV)NS基因特异性的脱氧核酶(DNAzyme,DZ-NS)在培养的人气道上皮细胞(9HTEo)体系内对甲型流感病毒复制的抑制效应.方法光镜观察DZ-NS对IFV致9HTEo细胞病变效应变化的影响;病毒空斑形成阻断试验及四甲基偶氮唑盐比色试验(MTT)检测脱氧核酶DZ-NS对IFV复制的抑制和对感染IFV的9HTEo细胞的保护作用;RT-PCR检测DZ-NS对IFV mRNA表达的抑制作用。结果DZ-NS可明显改善IFV所致的细胞病变并能明显提高细胞感染IFV后的存活率(P<0.01);可显著抑制IFV-NS基因mRNA的表达;空斑实验病毒抑制率可达80.11%;DZ-NS在细胞内对IFA病毒表达的抑制效率,明显高于作为对照的反义寡核苷酸(antisense oligonucleotides,AS-NS)(P<0.05)。结论在9HTEo感染IFV细胞模型系统,DZ-NS能高效阻断IFV的NS基因的表达,是一种特异性的、高效的抗IFV基因治疗剂。
Objective To investigate the inhibitory effect of DNAzyme (DZ-NS) specific to the NS gene of influenza virus Avian influenza virus (IFV) on the replication of influenza A virus in cultured human airway epithelial cells (9HTEo) The effect of DZ-NS on the pathological changes of 9HTEo cells induced by IFV was observed under light microscope. The inhibition of viral plaque formation and tetramethylzirconate colorimetric assay (MTT) were used to detect the inhibition of IFV replication by DZ-NS And protective effect on IFV-infected 9HTEo cells. The inhibitory effect of DZ-NS on IFV mRNA expression was detected by RT-PCR. Results DZ-NS could significantly improve the cytopathic effect induced by IFV and significantly increase the survival rate of cells infected with IFV (P <0.01), and significantly inhibit the expression of IFV-NS mRNA. The inhibition rate of plaque virus was 80.11 %. The inhibition efficiency of DZ-NS on IFA virus expression in cells was significantly higher than that of antisense oligonucleotides (AS-NS) (P <0.05). CONCLUSION: In the 9HTEo-infected IFV cell model system, DZ-NS can effectively block the expression of NS gene in IFV and is a specific and effective anti-IFV gene therapy agent.