摘要
目的 探讨脱氧核酶 (DNAzyme ,Deoxyribozyme)在培养细胞内对呼吸道合胞病毒 (RSV)复制的影响 ,为使用脱氧核酶控制RSV感染提供理论依据。方法 采用光镜、电镜观察自行设计与合成的抗RSV基因组RNA脱氧核酶DZ6 0 4对RSV致 9HTE细胞病变效应及超微结构变化的影响 ;病毒空斑形成阻断试验以及四甲基偶氮唑盐比色试验检测脱氧核酶DZ6 0 4对RSV复制的抑制和对RSV感染的 9HTE细胞的保护作用。结果 脱氧核酶DZ6 0 4可明显改善RSV所致的细胞病变 ,干预组病变出现时间和完全病变时间延迟 ,随着RSV感染量的逐渐减小 ,效果逐渐增强 ;脱氧核酶的病毒抑制效应与其浓度有关 ,浓度越高 ,效果越明显 ;脱氧核酶能明显减轻与推迟病毒感染早期细胞的超微结构变化 ;脱氧核酶DZ6 0 4具有抗RSV效应 ,能明显提高细胞感染RSV后的存活率 (P <0 0 0 1) ,非特异性脱氧核酶不具备抗RSV作用 (P >0 0 5 )。结论 我们设计与合成的抗RSV基因组RNA脱氧核酶DZ6 0 4在培养人气道上皮细胞内具有特异性抗RSV效应。
Objective DNAzyme/Deoxyribozyme is another novel molecular biological tool following the ribozyme. DNAzyme consists of a 15-nucleotide (nt) internal loop as its catalytic domain and two flanking substrate-recognition domains of 7 to 8 nt each which is complementary to substrate. The RNA substrate is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. DNAzyme has been applied in fields such as viral infectious disease, tumor, cardiovascular disease and genetic disease. But there is no report about using DNAzyme for anti-respiratory syncytial virus purpose. To observe the inhibitory effects of RNA-cleaving DNAzymes on respiratory syncytial virus (RSV) replication in cultured cells. Methods Anti-RSV RNA-cleaving DNAzyme DZ604 was designed to target the RSV genome at the start of the NS2 gene in an effort to inhibit the RNA replication. Microscope and electron microscope were used to observe the effects of anti-RSV genomic RNA DNAzyme on cytopathogenic effect (CPE) and ultrastructural change of 9HTE cell induced by RSV infection. Viral plaque forming reduction assay and MTT assay were used to detect the anti-RSV activity and protective function for RSV infected 9HTE cells of DNAzyme. Results Anti-RSV genomic RNA DNAzyme (DZ604)significantly improved CPE of RSV-infected 9HTE cells. The time to appearance of CPE and of total CPE was delayed by using DZ604 in a dose-dependant manner. At a 5 μmol /L concentration of DZ604, CPE of 9HTE cells induced by RSV infection at 10 and 1 multiplicity of infection (MOI) was not improved. At smaller MOI (0.1, 0.01, 0.001, 0.000 1) of RSV infection, CPE of 9HTE cells was significantly lightened by DZ604 at the same concentration. DZ604 also significantly improved ultrastructural change of 9HTE cells at early stage of RSV infection. Reduction in RSV yield was 85.56% and 8.33% at concentrations of 5 μmol/L and 0.25 μmol/L of DZ604. DZ604 inhibited RSV yield in a dose-dependent manner ( P <0.05). Non-specific DNAzyme did not have anti-RSV activity ( P> 0.05). Conclusion Anti-RSV genomic RNA DNAzyme designed and synthesized in our laboratory was capable of inhibiting respiratory syncytial virus replication specifically in cultured cells. Our data indicated that DNAzymes could be useful for the prevention against respiratory syncytial virus infection.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2003年第8期594-597,T003,共5页
Chinese Journal of Pediatrics
基金
国家自然科学基金资助项目 (3 0 0 70 3 3 1)
