沙门菌感染IPEC-J2细胞前后上清液外泌体鉴定及其m^(6)A相关蛋白表达和转录组分析

Identification of Exosomes from Supernatant of IPEC-J2 Cells Before and After Infected with Salmonella,and Expression and Transcriptome Analysis of m^(6)A Related Proteins

【目的】探究外泌体在沙门菌感染猪小肠上皮细胞(IPEC-J2细胞)过程中的作用及分子调控机制。【方法】利用间接免疫荧光试验观察沙门菌感染前后IPEC-J2细胞中沙门菌抗体分布水平,用超速离心法提取沙门菌感染IPEC-J2细胞上清液中的外泌体,并通过透射电镜观察外泌体形态、纳米颗粒跟踪分析测定外泌体粒径、Western blotting检测外泌体标记蛋白分子(CD9、CD63、CD81、TSG101)进行外泌体鉴定;利用实时荧光定量PCR和Western blotting检测N6-甲基腺嘌呤(m^(6)A)相关蛋白表达,同时结合转录组测序筛选上清液外泌体差异表达基因。【结果】间接免疫荧光试验结果显示,感染组IPEC-J2细胞中可观察到大量沙门菌分布,表明沙门菌可感染IPEC-J2细胞;透射电镜和外泌体粒径分析结果显示,IPEC-J2细胞分泌的外泌体为双层膜的囊泡,直径为30~150 nm;Western blotting结果显示,C-J2细胞分泌的外泌体存在CD9、CD63、CD81和TSG101特异性标记蛋白。沙门菌感染后外泌体中m^(6)A修饰相关基因表达检测发现,ALKBH 5、YTHDC 2及YTHDF 1基因的mRNA和蛋白表达均显著或极显著上调(P<0.05;P<0.01)。转录组测序结果显示,沙门菌感染IPEC-J2细胞前后外泌体中存在1524个差异表达基因,包括842个下调基因和682个上调基因。GO功能富集分析表明,沙门菌感染主要影响免疫系统过程、细胞连接和酶调节剂活性等生物过程;KEGG通路富集分析表明,其与IgA生产的肠道免疫网络、T细胞受体信号通路和Toll样受体信号通路等密切相关。【结论】本研究成功分离和鉴定了沙门菌感染IPEC-J2细胞上清液中的外泌体,并且表明外泌体中m^(6)A修饰可介导免疫信号通路来调控沙门菌感染宿主细胞,该研究可为今后深入探究外泌体对猪肠道沙门菌感染的分子机制提供理论参考。

【Objective】The aim of this study was to investigate the effect and molecular mechanism of exosomes in regulating the porcine small intestinal epithelial cells(IPEC-J2 cells)during Salmonella infection.【Method】The distribution of Salmonella antibody in IPEC-J2 cells after Salmonella infection was observed by indirect immunofluorescence assay.The exosomes in the cell supernatant of Salmonella-infected IPEC-J2 cells were observed by supercentrifugation.The morphology of the exosomes was observed by transmission electron microscopy(TEM),the particle size of the exosomes was measured by nanoparticle tracking analysis(NTA),and the molecular markers CD9,CD63,CD81 and TSG101 were detected by Western blotting.Additionally,the expression of N6-methyladenosine(m^(6)A)related proteins was detected by Real-time quantitative PCR and Western blotting.Besides,the differentially expressed genes of exosomes in supernatant were screened by transcriptome sequencing.【Result】Indirect immunofluorescence assay results showed that Salmonella adhered to IPEC-J2 cells,which indicated that Salmonella was able to infect IPEC-J2 cells.TEM and NTA analysis showed that the exosomes had bilayer membrane vesicles with a diameter of 30-150 nm,and four exosomal protein markers CD9,CD63,CD81 and TSG101 were expressed in the purified exosomes by Western blotting.The expression of m^(6)A modification-related genes and proteins ALKBH5,YTHDC2 and YTHDF1 was significantly or extremely significantly up-regulated in exosomes after Salmonella infection(P<0.05 or P<0.01).Transcriptome sequencing results showed that there were a total of 1524 differentially expressed genes(DEGs)in exosomes of IPEC-J2 cells after Salmonella infection,including 842 down-regulated genes and 682 up-regulated genes.Go function enrichment analysis showed that Salmonella infection mainly affected the immune system process,cell junction and enzyme regulator activity and other biological processes.KEGG pathway enrichment analysis revealed that differentially expressed genes were closely related to intestinal immune network for IgA production,T cell receptor signaling pathway,Toll-like receptor signaling pathway.【Conclusion】In this study,exosomes were successfully isolated and identified in the supernatant of Salmonella infected IPEC-J2 cells,and it indicated that m^(6)A modification could regulate Salmonella infection through the activation of immune signaling pathways,and this study could provide a reference for the subsequent study of exosome-induced release of exosomes from IPEC-J2.