禽呼肠孤病毒σA基因对鸡胚成纤维细胞天然免疫相关基因转录水平的调控影响

Regulation of avian reovirus σA gene on expression of innate immune genes in chicken embryo fibroblasts

为了明确禽呼肠孤病毒(Avian reovirus,ARV)σA基因对宿主天然免疫应答的影响,本研究利用实时荧光定量PCR方法检测pCAGEN-σA转染鸡胚成纤维细胞(DF1)后,不同时间点(转染后第3、6、12、24和36小时)DF1细胞中天然免疫相关基因,包括Toll样受体(TLRs)、MDA5、MAVS、My D88、TRIF、IRF-3、IRF7、NF-κB、IFN-α、IFN-β、IL-6、IL-8、IFITM3、Mx1和OASL等基因的转录水平变化。结果,与空载体转染细胞相比,pCAGEN-σA转染细胞中上述这些天然免疫基因的m RNA水平出现不同程度变化(P<0.05或P<0.01)。TLR3应对最快速,其m RNA水平在转染后第3小时和第6小时迅速上升,但9 h后逐渐下降;MDA5和TLR7的m RNA水平,则分别在转染后第6小时和第12小时开始显著升高,并分别在转染后第12小时和第36小时达到峰值(P<0.05或P<0.01);TLR15的m RNA水平整体变化不明显。My D88和MAVS的m RNA水平呈表达上调趋势;TRIF在转染后第6小时表达量迅速达到峰值(2.83倍),随后急速下降。NF-κB呈表达上调趋势;而IRF3/7的表达则被抑制。I型干扰素(IFN-α和IFN-β)呈现相似的表达模式,在转染后第6小时和第24小时表达量增加,其他时间点均为表达量下降; IL-6和IL-8则表达上调,分别在转染后第9小时和第6小时达到峰值(P<0.01)。IFITM3、Mx1、OASL的m RNA水平也显著升高,均在转染后第12小时达到峰值(P<0.01)。上述试验结果表明,在DF-1细胞中过表达σA基因,能显著引起天然免疫相关基因的表达的变化,为进一步阐释ARV的分子致病机制和免疫应答机理奠定了基础。

This study is aimed to assess the influences of avian reovirus(ARV)σA gene on expression of innate immune genes in chicken embryo fibroblasts cells.(Method)Using the quantitative real-time PCR assays,it was performed to determine the transcriptional levels of TLR3,TLR7,TLR15,MDA5,My D88,MAVS,TRIF,IRF3/7,NF-κB,IFN-α,IFN-β,IL-6,IL-8,IFITM3,Mx1 and OASL at 3 h,6 h,12 h,24 h and 36 h post-transfection in p CAGEN-σA-transfected cells.In result,the m RNA transcriptional levels of these innate immune-associated genes were significantly induced(P<0.05 or P<0.01).TLR3 were quickly up-regulated at 3 h and 6 h post-transfection, but gradually reduced at 9 h post-transfection.The m RNA transcriptional levels of MDA5 and TLR7 were obviously promoted at 6 h and 12 h post-transfection,and specially reached the summit maximum values at 12 h and 36 h post-transfection,respectively(P<0.05 or P<0.01);TLR15 did not exhibit obvious expression changes.The m RNA expression levels of My D88 and MAVS was up-regulated within 48 h.Meanwhile,the m RNA expression level of TRIF was peaked at 6 h post-transfection,but subsequently reduced after 12 h post-transfection.The m RNA expression levels of NF-κB were up-regulated during the whole experiment course,meanwhile the IRF3/7 m RNA expression level was inhibited.IFN-α and IFN-β were up-regulated at 3 h and 6 h post-transfection,but were decreased at other time points post-transfection.IL-6 and IL-8 were significantly up-regulated and peaked at 9 h and 24 h post-transfection,respectively(P<0.05 或 P<0.01).The m RNA expression levels of IFITM3,Mx1 and OASL were significantly up-regulated and peaked at 12 h post-transfection(P<0.01).In conclusion,ARV σA gene is able to modulate the increased m RNA expression of the innate immune genes,which would contribute to further understand ARV pathogenesis and host innate immune responses to ARV infection.