作 者：
毕栏;赵鹏宇;王天硕;于思雯;肖佳薇;郭东华
关键词：
坏死杆菌;外膜囊泡;NLRP3炎性小体;自噬
摘 要：
为探究坏死杆菌(Fn)外膜囊泡(OMV)对羊中性粒细胞自噬及NOD样受体蛋白3(NLRP3)炎性小体激活的影响,本研究提取并纯化Fn OMV,经透射电子显微镜观察并测定纯度后,以5 μg/mL的OMV刺激羊中性粒细胞1h、2h、4h、6 h,以未刺激的细胞作为对照组,经姬姆萨染色和倒置光学显微镜观察羊中性粒细胞形态.电镜观察可见OMV为球形囊泡状结构且纯度较高.姬姆萨染色结果显示,细胞为典型的杆状核和分叶核样中性粒细胞.光学显微镜观察结果显示,对照组羊中性粒细胞为圆形,而5 μg/mL OMV刺激组细胞出现细胞肿胀、体积变大变形的现象,且刺激2 h~6 h上述细胞变化更明显.采用荧光定量RT-PCR检测OMV刺激后的羊中性粒细胞中炎性小体相关基因 NLRP3、Caspase-1、GSDMD、IL-1β、IL-18,自噬相关基因 mTOR、Beclin 1、MAP1LC3B、P62mRNA的相对转录水平.结果显示,与对照组相比,OMV刺激1 h时GSDMDmRNA的相对转录水平极显著升高(P<0.0001),其他炎性小体相关基因mRNA的相对转录水平无显著变化;刺激2 h时GSDMDmRNA的相对转录水平显著下降(P<0.05),炎性小体相关基因NLRP3、Caspase-1、IL-1β、IL-18 mRNA的相对转录水平均显著升高(P<0.05);刺激4 h和6 h时GSDMD mRNA的相对转录平极显著下降(P<0.0001),NLRP3、iL-1βmRNA的相对转录水平均显著升高(P<0.05),Caspase-1、IL-18mRNA的相对转录水平均无显著变化.自噬相关基因mTOR、Beclin 1、MAP1LC3B、P62mRNA的相对转录水平在不同刺激时间后均显著升高(P<0.05).上述结果表明,5 μg/mL的Fn OMV刺激羊中性粒细胞2 h可激活NLRP3炎性小体;5 μg/mL的Fn OMV可在基因水平上启动羊中性粒细胞自噬并形成自噬小体.本研究为Fn的致病机制研究奠定了基础.
译 名：
Autophagy and NLRP3 inflammasome induced by Fusobacterium necrophorum OMV in sheep neutrophils
关键词：
Fusobacterium necrophorum%outer membrane vesicles%NLRP3 inflammasome%autophagy
摘 要：
To investigate the effects of outer membrane vesicle(OMV)of Fusobacterium necrophorum on autophagy and NOD-like receptor protein 3(NLRP3)inflammasome activation of sheep neutrophils.OMV was extracted and purified.After the purity of OMV was identified by transmission electron microscope,sheep neutrophils were treated with OMV at a concentration of 5μg/mnL for 1 hour,2 hours,4 hours and 6 hours.The sheep neutrophils was stained by Giemsa staining and observed under a inverted optical microscope.The results of Giemsa staining showed that the cells were neutrophils with typical rod-shaped and lobulated nuclei.The results of optical microscopy showed that the sheep neutrophils in the mock group were round,while the cells in the 5μg/mL OMV stimulation group showed cell swelling,enlargement and deformation.The relative transcription levels of mRNA for the inflammasome-related genes NLRP3,Caspase-1,GSDMD,IL-1β and IL-18,and autophagy-related genes mTOR,Beclin 1,MAP1LC3B and P62 in sheep neutrophils were detected by fluorescence quantitative PCR(qRT-PCR).The results of qRT-PCR showed that compared with the mock group,the relative transcription level of GSDMD mRNA was significantly increased at 1 hour(P<0.0001),and the relative transcription levels of other inflammasome-related genes were not significantly changed.The relative transcription level of GSDMD mRNA decreased significantly after stimulation for 2 hours(P<0.05),and the relative transcription levels of inflammasome-related genes NLRP3,Caspase-1,II-1 β and IL-18 mRNA increased significantly(P<0.05).After stimulation for 4 hours and 6 hours,the relative transcription level of GSDMD mRNA decreased significantly(P<0.0001),the relative transcription levels of NLRP3 and IL-1β mRNA increased significantly(P<0.05),and the relative transcription levels of Caspase-1 and IL-18 mRNA did not change significantly.The relative transcription levels of autophagy-related genes mTOR,Beclin 1,MAP1LC3B,and P62 mRNA were significantly up-regulated after different stimulation times(P<0.05).The above results showed that 2 hours stimulation with 5μg/mL OMV could activate NLRP3 inflammasome in sheep neutrophils.5μg/mL of OMV could initiate autophagy of sheep neutrophils at the genetic level and form autophagosomes.This study provides a reference for the study of the pathogenic mechanism of F.necrophorum.
