基于转录组分析猪β-防御素114在巨噬细胞中的免疫调节作用及机制  

Analysis of Immunomodulatory Effects and Mechanisms of Porcineβ⁃Defensin 114 in Macrophages Based on Transcriptome

作　　者：李林峰 黄升[1,2] 陈利 江山 黄金秀[1,2] 杨飞云 王贵学[4] 刘作华 苏国旗[1,2] LI Linfeng;HUANG Sheng;CHEN Li;JIANG Shan;HUANG Jinxiu;YANG Feiyun;WANG Guixue;LIU Zuohua;SU Guoqi(Chongqing Academy of Animal Science,Chongqing 402460,China;Key Laboratory of Pig Science,Ministry of Agriculture and Rural Affairs,Chongqing 402460,China;College of Animal Science and Technology,Southwest University,Chongqing 400715,China;College of Bioengineering,Chongqing University,Chongqing 400044,China)

机构地区：[1]重庆市畜牧科学院,重庆402460 [2]农业农村部养猪科学重点实验室,重庆402460 [3]西南大学动物科学技术学院,重庆400715 [4]重庆大学生物工程学院,重庆400044

出　　处：《动物营养学报》2023年第8期5359-5373,共15页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：重庆市科研机构绩效引导专项(cstc2021jxjl80001);重庆市博士后基金特别资助项目(21309);重庆英才计划“包干制”项目(21201)。

摘　　要：本试验旨在基于转录组研究猪β-防御素114(PBD114)在巨噬细胞中的免疫调节作用及机制。试验采用不同浓度(1~256μg/mL)的PBD114处理猪肺泡巨噬细胞3D4/21,并分别在1、3、6和12 h收集细胞培养液,采用酶联免疫吸附试验(ELISA)法检测肿瘤坏死因子-α(TNF-α)和白细胞介素-10(IL-10)。为更加深入揭示PBD114在巨噬细胞中的免疫调节作用及机制,本试验通过转录组分析0(MOCK组)和100μg/mL PBD114(PBD114组)对培养12 h猪肺泡巨噬细胞3D4/21相关基因表达的影响,并采用实时荧光定量PCR(qRT-PCR)验证转录组测序结果。结果表明:1)适宜浓度PBD114能够显著促进猪肺泡巨噬细胞3D4/21分泌TNF-α和IL-10(P<0.05),并且存在剂量依赖和时间效应。2)转录组测序表明,MOCK组和PBD114组共鉴定出1 894个差异表达基因(DEGs)。其中,与MOCK组相比,PBD114组CXC基序趋化因子配体2(CXCL2)、白细胞介素-1β(IL-1β)、集落刺激因子3(CSF3)、白血病抑制因子(LIF)和白细胞介素-1α(IL-1α)等1 170个基因显著上调[差异倍数(FC)≥2,校正P值(adjusted P-value)≤0.001],B细胞淋巴瘤6β(Bcl6β)、B细胞淋巴瘤3(Bcl3)、CD2、CD4和CD83等724个基因显著下调(FC≥2,adjusted P-value≤0.001)。DEGs基因本体(GO)功能注释主要分布在分子功能、细胞组分和生物过程,DEGs数量分别是1 832、1 844和1 802个;京都基因与基因组百科全书(KEGG)通路显著富集到95条(Q<0.05),其中肿瘤坏死因子(TNF)信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、核因子-κB(NF-κB)信号通路、Toll样受体(TLR)信号通路和白细胞介素-17(IL-17)信号通路等信号通路参与免疫激活,DEGs数量分别是38、66、33、29和27个;蛋白质-蛋白质互作(PPI)和关键驱动分析(KDA)分析表明,RELB、TNF-α诱导蛋白3(TNFAIP3)和TNF受体相关因子1(TRAF1)等基因在PBD114调节巨噬细胞免疫中起着关键作用。3)qRT-PCR验证结果表明,8个随机选择的DEGs的表达趋势与转录组This experiment was conducted to investigate the immunomodulatory effects and mechanisms of por⁃cineβ⁃defensin 114(PBD114)in macrophages based on transcriptome.Porcine alveolar macrophages 3D4/21 were treated with PBD114 at different concentrations(1 to 256μg/mL),and cell cultures were collected at 1,3,6 and 12 h,respectively.Then the concentrations of tumor necrosis factor⁃α(TNF⁃α)and interleukin⁃10(IL⁃10)were detected by enzyme⁃linked immunosorbent assay(ELISA).In order to further reveal the immu⁃nomodulatory effects and mechanisms of PBD114 in macrophages,transcriptome was used to analyze the effects of 0(MOCK group)and 100μg/mL PBD114(PBD114 group)on related gene expression in porcine alveolar macrophages 3D4/21 cultured for 12 h.Real⁃time quantitative fluorescence PCR(qRT⁃PCR)was used to verify the transcriptome sequencing results.The results showed as follows:1)the appropriate concen⁃tration of PBD114 could significantly promote the secretion of TNF⁃αand IL⁃10 in porcine alveolar macropha⁃ges 3D4/21(P<0.05),and there were dose⁃dependent and time⁃dependent effects.2)Transcriptome sequen⁃cing showed that a total of 1894 differentially expressed genes(DEGs)were identified between MOCK group and PBD114 group.Compared with MOCK group,1170 genes such as CXC motif chemokine ligand 2(CX⁃CL2),interleukin⁃1β(IL⁃1β),colony stimulating factor 3(CSF3),leukaemia inhibitory factor(LIF)and interleukin⁃1α(IL⁃1α)in PBD114 group were significantly up⁃regulated[fold change(FC)≥2,adjusted P⁃value≤0.001],while 724 genes such as B⁃cell lymphoma 6β(Bcl6β),B⁃cell lymphoma 3(Bcl3),CD2,CD4 and CD83 were significantly down⁃regulated(FC≥2,adjusted P⁃value≤0.001).Gene ontology(GO)function annotations of DEGs were mainly distributed in molecular function,cell component and biological process,and the number of DEGs was 1832,1844 and 1802,respectively.The Kyoto encyclopedia of genes and genomes(KEGG)pathway was significantly enriched to 95 pathways(Q<0.05),in which

关 键 词：猪β-防御素114 巨噬细胞 免疫 炎症 

分 类 号：S811[农业科学—畜牧学]