反义硫代修饰寡核苷酸体外抗甲型流感病毒感染的效应研究

Study on antisense phosphorothioate oligodeoxynucleotide resistance to influenza A virus infection in vitro

目的在感染甲型流感病毒（influenza Avirus，IFAV）的人气道上皮细胞（9HTEO）中观察与IFAV PB1、M2、NS、PB2、HA基因互补的反义硫代修饰寡核苷酸（ASODN）的抗病毒活性。方法细胞外体系观察设计的ASODN效率，筛选对IFAV有效的3个ASODN进行9HTEO的抗病毒效应。倒置显微镜下观察IFAV的致细胞病变作用和ASODN的细胞保护作用。MTT方法测细胞存活率，空斑试验、RT．PCR、Westernblot、免疫荧光检测ASODN特异性抗病毒效应，MTF法检测ASODN对细胞的保护作用。结果IFAV感染复数（MO!）在0．1以上时，培养5d后IFAV感染的9HTEO细胞全部死亡。设计的ASODN能够提高感染细胞的存活率，且呈量效依赖关系。空斑试验、RT．PCR、Westernblot、免疫荧光试验证实在细胞水平、基因转录水平、蛋白水平针对IFAV PB1、M2、NS基因mRNA转录起始点保守序列设计的ASODN具有特异性抗IFAV作用。结论ASODN具有较明显的抗病毒活性，为进一步研究和开发新型抗IFAV药物提供了实验依据。为高致病性禽流感（H5N1）的基因治疗提供了新的思路和理论依据。

Objective To investigate the antiviral effects of antisense phosphorothioate oligodeoxynucleotide （ASODN）in 9HTEO infected with influenza A virus （IFAV） in vitro. Methods The ASODN which complemented to genomic PB1, M2, NS, PB2, HA mRNA of IFAV was used to investigate antiviral effection in vitro. The cytopathic effect （CPE） was observed, and the cell survival rates were measured by MTT assay, plaque assay, RT-PCR, Western blot and Immunofluorescence were performed to test anti-viral efficiency of PB1, M2, NS in cells mRNA and protein level. Results 9HTEO cells infected with IFAV almost all died when the multipicity of infection （MOI） is aboved after 5 days cell culture. The ASODN could increase the cell survival rates. The IFAV PB1, M2, NS significantly reduced CPE of IFAV infected 9HTEO cells, reduced the viral replication of IFAV in the cells （ P 〈 0.05）. Conclusion The ASODN which targeted the mRNA of IFAV gene showed a significant and specific anti-IFAV effect both in mRNA and protein level in cells culture system. The study indicates that the PB1, M2 and NS mRNA may play an important role in regulating IFAV replication, and ASODN may have inhibitory activity on IFAV replication. The results established the basis for further study on new drugs against IFAV infection include the highly pathogenic H5N1 influenza virus.