体外建立dsRNA结合甲基化转移酶抑制剂转染猪肾细胞的模型

摘要

Viral-diseases cause great economic losses in the livestock industry all over the world.Porcine RNA viruses produced doubled stranded RNA (dsRNA) as viral replication median,which induced type Ⅰ interferon,and inhibit the replication of viruses.That is more importance of host anti to RNA viral infection.Porcine dsRNA has chemotaxis effect on epithelial cells.DNA methyltransfereases inhibitor can induce the apoptosis and anti-viral replication.Using RT-PCR method,the experiment analyzed the relative expression of TLR3,IRF,.IFNA,and 50％ cell survive rate was analysis by CCK8 method.Finally,the assay build the ideal model of PK15 cells treated with Aza-CdR after transfected by Poly(Ⅰ:C).The model can effectively mimic the infection of alive viruses in vitro,which can be used in the routine laboratoy.The model of Poly(Ⅰ∶C) combined with Aza-CdR can be the immune stimulation to explain the relationship between host cells and alive viruses.%猪病毒性疾病对现代养猪业造成了巨大的经济损失.猪RNA病毒基因组在复制过程中形成双链RNA(dsRNA)的复制型中间体,并诱导Ⅰ型干扰素的产生,从而对病毒的增殖起抑制作用,这是宿主抗RNA病毒感染的重要天然防线.猪许多病毒的dsRNA对上皮细胞有明显的趋化作用,而DNA甲基化转移酶抑制剂Aza-CdR可诱导感染细胞凋亡并发挥抗病毒作用.本实验结合荧光定量PCR,计算干扰素相关基因(TLR3、IRF、IFNA)的相对表达量.同时,通过设计剂量和时间依赖实验,利用CCK8实验方法,计算细胞生存比率,最终确定dsRNA模拟物Poly I:C体外转染PK15细胞后,Aza-CdR处理的理想细胞转染模型.此模型能有效地在体外模拟活病毒感染细胞的效果,更便于在常规实验室使用,是研究猪宿主与病毒感染关系的理想细胞模型.