基于猪小肠上皮细胞炎症模型分析产肠毒素大肠杆菌诱导的转录组表达变化

doi:10.16431/j.cnki.1671-7236.2022.12.004

摘要/Abstract

摘要： 【目的】分析产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）诱导猪小肠上皮细胞（IPEC-J2）炎症反应的主要宿主调控基因及相关分子通路，为进一步揭示ETEC感染引发炎症反应的作用机制提供理论依据。【方法】用感染复数（multiplicity of infection，MOI）为100的ETEC菌液感染IPEC-J2细胞，18 h后收集细胞上清，通过ELISA方法检测炎性因子白细胞介素1β（interleukin 1 β，IL-1β）的分泌水平。由Illumina PE150测序平台进行高通量转录组测序，通过edgeR v 3.12.1软件对测序结果进行差异表达分析，利用GOseq和KOBAS 2.0软件对得到的差异表达mRNA进行GO功能和KEGG通路富集分析，随机挑选5条差异表达mRNA，利用实时荧光定量PCR对其验证。【结果】试验成功建立了ETEC感染诱导的IPEC-J2细胞炎症模型。与对照组相比，炎症模型组共发现529条差异表达mRNA，其中236条表达上调，293条表达下调，包括HSP90AA1、CGNL1、CADPS2、EHBP1等免疫相关基因。GO功能分析表明，ETEC感染后差异表达mRNA显著富集于细胞组分和生物过程，包括细胞内成分（intracellular part）、细胞质（cytoplasm）、核成分（nuclear part）、胞内膜结合细胞器（intracellular membrance-bounded organelle）、膜结合细胞器（membrance-bounded orgabelle）及细胞进程（cellular process）等。KEGG通路分析表明，炎症模型中差异表达基因大多数被注释到疾病相关通路及Toll样受体信号通路、mTOR信号通路、细胞周期、黏附连接等免疫相关信号通路。利用实时荧光定量PCR验证随机挑选的5条差异表达mRNA，结果与测序报告中差异表达mRNA变化趋势一致，证明测序结果可信。【结论】HSP90AA1、CGNL1、CADPS2和EHBP1等基因可能在ETEC黏附IPEC-J2细胞并诱导炎症反应中发挥关键作用，并通过Toll样受体、mTOR等信号通路进行免疫调控。研究结果为进一步揭示ETEC感染诱导炎症反应的分子机制及相关调控网络提供参考依据。

关键词: 产肠毒素大肠杆菌(ETEC); 猪小肠上皮细胞(IPEC-J2); 炎症模型; 转录组

Abstract: 【Objective】 This study was aimed to analyze the key host-regulated genes and related molecular pathways in the inflammatory response induced by enterotoxigenic Escherichia coli (ETEC) in porcine small intestinal epithelial cells (IPEC-J2), in order to provide theoretical basis for the investigation of inflammatory mechanism induced by ETEC infection.【Method】 The IPEC-J2 cells were infected with ETEC bacterial solution with a multiplicity of infection (MOI) of 100, and the medium supernatant was collected at 18 h post infection.The secretion of proinflammatory factor interleukin 1β (IL-1β) was detected by ELISA method.High throughput transcriptome sequencing was performed by Illumina PE150 sequencing platform.Differentially expressed analysis was performed on the sequencing results by edgeR v 3.12.1 software.GO function and KEGG pathway enrichment analysis were performed on the differentially expressed mRNA obtained by GOseq and KOBAS 2.0 softwares.Five differentially expressed mRNA were randomly selected and verified by Real-time quantitative PCR.【Result】 A model of ETEC infection-induced inflammation in IPEC-J2 cells was successfully established.Compared with control group, a total of 529 differentially expressed mRNA were identified in inflammatory model group, of which 236 were up-regulated and 293 were down-regulated, including immune-related genes such as HSP90AA1, CGNL1, CADPS2 and EHBP1.GO function analysis showed that differentially expressed mRNA after ETEC infection were significantly enriched in cellular component and biological process, such as the intracellular part, cytoplasm, nuclear part, intracellular membrance-bound organelle, membrance-bound orgabelle and cellular process.KEGG pathway analysis showed that most of differentially expressed genes after ETEC infection were annotated as disease-related pathways as well as immune-related signaling pathways such as Toll-like receptor signaling pathway, mTOR signaling pathway, cell cycle and adhesion junctions.Real-time quantitative PCR was used to validate the expression of five randomly selected differentially expressed mRNA, the results were consistent with the trend of differentially expressed mRNA in the sequencing result, which indicated that the sequencing results were reliable.【Conclusion】 HSP90AA1, CGNL1, CADPS2 and EHBP1 genes might play a key role in ETEC adhesion to IPEC-J2 cells and induce inflammatory responses, and immunoregulated through Toll-like receptors, mTOR and other signaling pathways.The results provided a reference for further understanding the molecular mechanisms and regulatory networks involved in the inflammatory response induced by ETEC infection.

Key words: enterotoxigenic Escherichia coli (ETEC); porcine small intestinal epithelial cells (IPEC-J2); inflammation model; transcriptome

中图分类号:

S852.61⁺2

朱悦, 刘迎迎, 李莹慧, 李培, 朱凯晴, 贾垌, 李妍. 基于猪小肠上皮细胞炎症模型分析产肠毒素大肠杆菌诱导的转录组表达变化[J]. 中国畜牧兽医, 2022, 49(12): 4545-4553.

ZHU Yue, LIU Yingying, LI Yinghui, LI Pei, ZHU Kaiqing, JIA Tong, LI Yan. Analysis of Enterotoxigenic Escherichia coli Induced Transcriptomic Changes of Porcine Intestinal Epithelial Cell Based on the Inflammatory Model[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4545-4553.

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