不同生长期坏死杆菌OMVs的分离鉴定及其对RAW264.7细胞分泌TNF-α、IL-8的影响

Isolation and Identification of Fusobacterium necrophorum OMVs at Different Growth Phase and its Effect on the Secretion of TNF-αand IL-8 in RAW264.7 Cells

为初步探究坏死杆菌不同生长期外膜囊泡(OMVs)在形态、蛋白组成和功能的差异,利用密度梯度离心法提取坏死杆菌OMVs,通过透射电镜观察OMVs的形态结构,SDS-PAGE和Western blot分析OMVs的蛋白组成差异,ELISA检测OMVs刺激RAW264.7细胞后TNF-α和IL-8的分泌情况。结果显示:坏死杆菌在培养2 h后进入对数生长期,在8 h时达到峰值,随后进入稳定期和衰亡期;坏死杆菌不同生长期均可产生球形的OMVs,在坏死杆菌培养24 h时产生的OMVs中43 K OMP和白细胞毒素的水平显著高于其他各组(P<0.01);坏死杆菌培养12 h时产生的OMVs刺激RAW264.7细胞12 h和24 h后分泌的IL-8和TNF-α水平均高于其他组。因此,坏死杆菌稳定期产生的OMVs可以更好的刺激RAW264.7细胞产生炎症因子IL-8和TNF-α。

To investigate the morphology,protein composition,and function of outer membrane vesicles(OMVs)in Fusobacterium necrophorum throughout the growth phase,F.necrophorum OMVs were isolated and purified using gradient centrifugation.A transmission electron microscope was used to analyze the morphology of OMVs(TEM).SDS-PAGE and Western blot were used to analyze the protein compositions.After stimulation with OMVs,ELISA was used to measure the levels of IL-8 and TNF-αin the cells.The results showed that after 2 hours of culture,F.necrophorum entered the logarithmic phase,peaked at 8 hours,and then entered the stationary phase and decline phase.The levels of 43K OMP and leukotoxin in OMVs produced by F.necrophorum cultured for 24 hours were significantly higher than those in other groups(P<0.01).OMVs produced by F.necrophorum cultured for 12 hours stimulated RAW264.7 cells secreted IL-8 and TNF-αlevel after 12 and 24 hours was the highest.As a result,OMVs produced by F.necrophorum stable phase could better stimulate RAW264.7 cells to produce inflammatory factors IL-8 and TNF-α.