牛坏死杆菌对EPH4细胞的增殖抑制和诱导凋亡作用

Inhibition of Proliferation and Induction of Apoptosis of EPH4 Cells by Fusobacterium necrophorum

为探究牛坏死杆菌对小鼠乳腺上皮细胞系(EPH4细胞)增殖和凋亡的影响,本研究将坏死杆菌(牛A25菌株)以感染复数(MOI)为100感染EPH4细胞,用EdU细胞增殖法检测细胞增殖率,用Hoechst染色以及DNA Ladder检测细胞凋亡,并用实时荧光定量PCR(RT-qPCR)检测凋亡基因的mRNA相对表达量变化。EdU检测结果显示,坏死杆菌可抑制EPH4细胞增殖;Hoechst染色以及DNA Ladder检测结果均显示,坏死杆菌可促进细胞凋亡的发生;RT-qPCR结果显示,与对照组相比,坏死杆菌感染EPH4细胞2 h时,促凋亡基因Bax、Bax/Bcl-2的mRNA相对表达量显著上调(P<0.05),感染4 h时,促凋亡基因Caspase-3和Caspase-9的mRNA相对表达量极显著上调(P<0.001),感染6 h时,促凋亡基因AIF的mRNA相对表达量极显著上调(P<0.001)。由此可见,牛坏死杆菌能够抑制EPH4细胞增殖且诱导细胞凋亡,为进一步研究坏死杆菌致病机制提供了相关数据支持。

To investigate the effects of Fusobacterium necrophorum on the proliferation and apoptosis of mouse mammary epithelial cell line(EPH4 cells),this study infected EPH4 cells with F.necrophorum(strain A25)at a multiplicity of infection(MOI)of 100 and detected the cell proliferation rate by EdU cell proliferation assay,the apoptosis by Hoechst staining and DNA Ladder,and the mRNA relative expression of apoptotic genes by real-time fluorescence quantitative PCR(RT-qPCR).The results of EdU assay showed that F.necrophorum inhibited the proliferation of EPH4 cells.Hoechst staining and DNA Ladder showed that F.necrophorum promoted apoptosis.The RT-qPCR results showed that compared with the control group,the mRNA relative expression levels of pro-apoptotic genes Bax and Bax/Bcl-2 were significantly upregulated when EPH4 cells were infected with F.necrophorum for 2 h(P<0.05),the mRNA relative expression levels of pro-apoptotic genes Caspase-3 and Caspase-9 were significantly up-regulated at 4 h after infection(P<0.001),and the mRNA relative expression levels of pro-apoptotic gene AIF were significantly up-regulated at 6 h after infection(P<0.001).Thus,F.necrophorum could inhibit proliferation and induce apoptosis of EPH4 cells,which underline the pathogenesis of F.necrophorum infection.