反义硫代脱氧寡核苷酸体外抑制CVB持续感染的研究  

Study on inhibition of persistent infection of Coxsackievirus B group by antisense phosphorothioate oligodeoxynucleotide in vitro

作　　者：苏琦华[1] 訾自强[1] 张霞[1] 

机构地区：[1]天津医科大学微生物学教研室,天津300070

出　　处：《天津医科大学学报》2008年第3期275-278,共4页Journal of Tianjin Medical University

基　　金：天津市自然科学基金资助项目(013613111);天津市卫生局科学基金资助项目(089387462)

摘　　要：目的:了解21聚体反义硫代脱氧寡核苷酸(AODN)体外对CVB3、CVB5持续感染的抑制作用。方法:人工合成AODN,进行脂质体包埋,并按不同浓度分别加入CVB3-ECV304、CVB5-ECV304持续感染细胞模型。对药物作用后持续感染细胞模型,观察其培养上清液致Vero细胞病变能力的改变;ELISA法检测CVB3、CVB5抗原产生的抑制率;RT-PCR检测细胞内病毒RNA表达情况;ELISA检测培养上清液分泌TNF-α浓度。同时进行正义和随机寡核苷酸(SODN、RODN)抗病毒能力检测,并与AODN结果比较。结果:AODN可抑制持续感染CVB3、CVB5抗原合成,随药物浓度的增高,抗原抑制率也升高;经AODN作用后,持续感染细胞培养上清液致Vero细胞病变能力下降,上清液中病毒滴度减少;RT-PCR在经AODN作用的CVB3-ECV304、CVB5-ECV304组中未检测到病毒RNA;经AODN作用后,持续感染的CVB3-ECV304、CVB5-ECV304细胞产生TNF-α浓度升高;针对CVB35′端非编码区基因组序列合成的AODN对CVB5持续感染也有较强的抑制作用;AODN对持续感染病毒的抑制作用强于SODN和RODN组。结论:AODN能体外抑制CVB3、CVB5持续感染,刺激细胞合成TNF-α。这种作用有序列特异性,但同组间病毒有交叉抑制作用,这种作用可能与CVB3、CVB5基因序列同源有关。Objective： To study the inhibition of CVB3 and CVB5 in persistent infection by 21 mer antisense phosphorothioate oligodeoxynucleotide （AODN） in vitro. Methods：AODN was synthesized and wrapped by Lipofectamine regeant. This AODN was transfected with different concentration to CVB3- ECV304 and CVB5-ECV304 cell models. The inhibitory effects of AODN were evaluated with a number of indexes, including inhibition of CPE on Vero cells caused by supernanant of cell models, inhibitory rates of CVB3 and CVB5 antigens by ELISA, viral RNA expression inside the cell models by RT-PCR and the concentration of TNF-α in cell model＇s supernanant by ELISA. At the same time, the effects of sense oligodeoxynucleotide （SODN） and randomized nonsense-sequence oligodeoxynucleotide （RODN） were detected and compared with AODN. Results：The specific AODN decreased the production of CVB3 and CVB5 antigens in persistent infection cell models and this effect depended on the AODN＇ s dose. After reacted by AODN, the degree of CPE on Vero cells caused by supernanat of cell models was inhibited. The viral titers in culture supertanant decreased and viral RNA couldn＇t be detected by RT-PCR. The concentration of TNF-α of cell models increased. ADON which was synthesized according to genes of CVB3 also had strongly inhibited to CVB5 persistent infection. The inhibition of AODN was stronger than SODN and RODN. Conclution： AODN can inhibit persistent infections of CVB3 and CVB5 in vitro and induce cells to produce TNF-α. This effect has sense specific but there is cross inhibition between the same viral groups. Maybe it is related to the homogenous genetic sense of CVB3 and CVB5.

关 键 词：持续感染 柯萨奇B组病毒 反义硫代脱氧寡核苷酸 

分 类 号：R373[医药卫生—病原生物学] R542.21[医药卫生—基础医学]