摘要
为了探讨宿主巨噬细胞(J774A.1细胞)在猪血凝性脑脊髓炎病毒(Procine hemagglutinatin encephalomyelitis virus,PHEV)感染过程中的作用及其参与炎性反应的应答特征,本研究通过CPE观察、TCID50、免疫荧光(IFA)、ELISA和PT-PCR等方法,对PHEV感染J774A.1细胞的增殖特性及炎性因子IL-6、TNF-a、IL-1β等mRNA转录水平和蛋白水平的表达情况进行检测分析。IFA的检测结果显示,感染后8h后仅有少量的被荧光抗体标记细胞出现绿色荧光,随着感染时间的延长,荧光标记的细胞逐渐增加,直至感染后32~40h出现大量细胞出现荧光。TCID50结果显示,PHEV感染滴度的增值趋势与mRNA含量的变化基本一致,在感染后24~32h增殖速度最快,至32h病毒感染滴度达到最高。荧光定量RT-PCR结果显示,PHEV感染可诱导J774A.1细胞分泌的TNF-a、IL-6、IL-1βmRNA转录水平均显著升高,尤其IL-6mRNA的表达量显著增加,在48h达到对照组的25倍;TNF-αmRNA的表达量在感染后8h内增加为对照组的4倍,随后一直维持较高水平。ELISA结果显示,TNF-a、IL-6、IL-1β蛋白水平表达也显著增加。上述研究结果表明,PHEV不仅可以感染J774A.1细胞,而且能诱导其分泌多种细胞因子参与免疫应答,为进一步阐明机体重要免疫细胞在PHEV过程中的作用奠定了基础。
In order to explore the role of procine hemagglutinatin encephalomyelitis virus in macrophages(J774A.1)and the mechanism for cellular inflammatory responses between the host and the virus,the level of inflammatory cytokine mRNA and protein expression including the TNF-a,IL-6and IL-1βafter infected in different times was tested by ELISA,PT-PCR.The mon-olayer cells of macrophages(J774A.1cell lines)were infected with HEV-67 Nin vitro to analyze proliferation characteristics by CPE observation,TCID50 immunofluorescence assay(IFA)and other measuring methods.IFA results showed that viral antigen could be detected at 8hp.i,and most of cells were infected between 32-40 hp.i.The results showed that,the mRNA transcript levels of TNF-a,IL-6,IL-1βwere significantly increased p.i.The mRNA transcript levels of IL-6were 25 times higher than the control group,at 48 hp.i.The mRNA transcript levels of TNF-a and IL-1βwere persistently increased from 8hp.i.The protein expression levels of TNF-a,IL-6and IL-1βwere also rapidly increased.The results confirmed that the inflammation cytokines mRNA and protein expression level would significantly increase after infected macrophages by PHEV.PHEV not only infects macrophages,but also induces the secretion of various cytokines involved in the immune response,which laid a foundation for further study of the role of immune cells in the PHEV infection.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第5期680-685,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31172291
31272530
31472194)
吉林省自然科学基金资助项目(20130101106JC)
