作 者：
王天硕;于思雯;毕栏;赵鹏宇;蒋凯;肖佳薇;郭东华
单 位：
;黑龙江八一农垦大学动物科技学院农业农村部东北寒区牛病防治重点实验室
摘 要：
为探究牛坏死杆菌对小鼠巨噬细胞系(RAW264.7细胞)增殖和自噬的影响,本研究将牛坏死杆菌A25株以MOI 10感染RAW264.7细胞作为实验组,以未感染的RAW264.7细胞作为对照组.分别于感染后1 h、2 h、4 h收集细胞,采用Ed U和MDC染色法观察细胞荧光,并计算各组细胞增殖率及细胞自噬小体的数量.Ed U染色法观察可见,各实验组随着感染时间的延长,绿色荧光的量逐渐减少,对照细胞绿色荧光的量基本无变化,且与对照组相比,随着感染时间的延长,感染组细胞增殖率均极显著降低(P<0.05);MDC染色法观察结果可见,与对照组相比,随着感染时间的延长,感染组细胞中的绿色荧光逐渐增强,且随着感染时间的延长,细胞中自噬小体的数量与对照组相比均极显著增多(P<0.01).上述结果表明,牛坏死杆菌感染RAW264.7细胞后抑制细胞的增殖,并且诱导该细胞发生自噬.分别采用荧光定量PCR(RT-qPCR)和western blot检测上述各组细胞中自噬基因Beclin-1和P62 mRNA的相对转录水平及自噬蛋白LC3和P62的相对表达水平.RT-q PCR结果显示,与对照组相比,感染1 h后RAW264.7细胞中Beclin-1基因mRNA的相对转录水平无显著变化;感染2 h和4 h时该基因mRNA的相对转录水平极显著升高(P<0.01),P62基因mRNA的相对转录水平在不同感染时间均极显著降低(P<0.001);western blot结果显示,与对照组相比,各实验组细胞中LC3-Ⅱ蛋白的表达量于不同感染时间均极显著升高(P<0.01),P62蛋白的表达量于不同感染时间均极显著降低(P<0.001).上述结果从自噬相关基因mRNA转录水平和其蛋白表达水平均证明牛坏死杆菌诱导RAW264.7细胞发生了自噬,首次证实牛坏死杆菌可以诱导RAW264.7细胞发生自噬,并且抑制该细胞的增殖,本研究为进一步探究牛坏死杆菌的感染机制奠定实验基础.
译 名：
Preliminary exploration on the effect of Fusobacterium necrophorum on the proliferation and autophagy to mouse macrophages
单 位：
College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Key Laboratory of Bovine Disease Control in Northeast China Ministry of Agriculture and Rural affairs,Daqing 163000,China
关键词：
Fusobacterium necrophorum%RAW264.7%autophagy
摘 要：
To investigate the effects of Fusobacterium necrophorum on the proliferation and autophagy of mouse macrophage cell lines(RAW264.7 cells),the RAW264.7 cells were infected with Fusobacterium necrophorum(strain A25)at a multiplicity of infection(MOI)of 10 for 1 hour,2 hours and 4 hours as experimental group,RAW264.7 cells without any treatment were used as the control group.The cell fluorescence was observed by EdU and MDC staining,and the cell proliferation rate and the number of autophagosomes in each group were calculated.EdU staining showed that the amount of green fluorescence decreased gradually with the prolongation of infection time,but control cells'green fluorescence did not change.Compared with the control group,the cell proliferation rate of the infection group significantly reduced with the prolongation of infection time(P<0.05).The results of MDC staining showed that compared with the control group,the green fluorescence gradually increased with the prolongation of infection time in the infection group,and the number of autophagosomes increased significantly with the prolongation of infection time compared with the control group.The above results showed that the infection of RAW264.7 cells by Fusobacterium necrophorum inhibited its proliferation and induced autophagy.The transcription levels of autophagy genes Beclin-1 and P62 and the relative expression levels of autophagy proteins LC3 and P62 in the above groups were detected by fluorescence quantitative PCR(RT-qPCR)and western blot.RT-qPCR results showed no significant change in the relative mRNA transcription level of the Beclin-1 gene at 1 hour after infection compared with the control group.The relative mRNA transcription level of this gene increased significantly at 2 hours and 4 hours after infection(P<0.01),and the relative mRNA transcription level of P62 gene was significantly down-regulated at different infection times(P<0.001).Western blot results showed that compared with the control group,the expression of LC3-Ⅱprotein was significantly increased at different infection times(P<0.01),and the expression of P62 protein was significantly decreased at different infection times(P<0.001).The above results further demonstrated that Fusobacterium necrophorum induced autophagy in RAW264.7 cells at the mRNA and protein expression levels of autophagy-related genes.This is also the first time that Fusobacterium necrophorum can induce autophagy in RAW264.7 cells.This study provides an experimental basis for exploring the infection mechanism of Fusobacterium necrophorum.
