猪圆环病毒2型ORF9基因对PK-15细胞的影响

Effect of ORF9gene of porcine circovirus type 2on PK-15cells

为探究猪圆环病毒2型(PCV2)ORF9基因对猪肾细胞(PK-15)的影响,通过构建ORF9基因真核表达质粒,并转染至PK-15细胞中,采用流式细胞术及qRT-PCR方法检测过表达ORF9基因对PK-15增殖、凋亡、免疫的影响。结果显示,PCV2ORF9基因过表达可显著增加内质网应激标志基因GRP78的表达水平;增加PK-15细胞S期细胞数,使细胞增殖速率增快;增加PK-15细胞凋亡率,上调凋亡相关基因caspase-3、caspase-8、caspase-9、p53和Bax的表达水平(P<0.01),下调凋亡相关基因Bcl-2的表达水平;上调免疫相关基因IL-8、IL-10、NF-κB、TNF-α的表达水平(P<0.01),下调免疫相关基因IL-2、IFN-β、IL-12的表达水平(P<0.01)。结果表明,PCV2ORF9基因可能促进PK-15细胞增殖与凋亡,并在PK-15细胞逃逸过程中发挥一定作用。

To investigate the effects of ORF9gene of porcine circovirus type 2(PCV2)on PK-15,eukaryotic expression plasmid was constructed and transfected into PK-15cells,and the effects of overexpression of ORF9on proliferation,apoptosis and immunization of PK-15cells were examined by flow cytometry and qRT-PCR.The results showed that ORF9gene overexpression significantly up-regulated the expression levels of the ER stress marker gene GRP78,increased the number of S phase cells,accelerated cell cycle progression,increased the apoptosis rate of PK-15cells,up-regulated the expression levels of apoptosis-related genes caspase-3,caspase-8,caspase-9,p53 and Bax(P<0.01),down-regulated the expression levels of apoptosis-related genes Bcl-2,up-regulated the expression levels of immune-related genes IL-8,IL-10,NF-κB and TNF-α(P<0.01),and down-regulated the expression levels of immune-related genes IL-2,IFN-βand IL-12(P<0.01).The above results indicate that ORF9gene may promote the proliferation and apoptosis of PK-15cells and play a role in the escape process of PK-15cells.