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绵羊肺炎支原体感染对绵羊肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7蛋白质组的影响

张颖 张凯 马金成 李小杰 马春骥 高力扬

张颖,张凯,马金成,等. 绵羊肺炎支原体感染对绵羊肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7蛋白质组的影响 [J]. 福建农业学报,2020,35(11):1244−1251 doi: 10.19303/j.issn.1008-0384.2020.11.010
引用本文: 张颖,张凯,马金成,等. 绵羊肺炎支原体感染对绵羊肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7蛋白质组的影响 [J]. 福建农业学报,2020,35(11):1244−1251 doi: 10.19303/j.issn.1008-0384.2020.11.010
ZHANG Y, ZHANG K, MA J, et al. Proteomes of Sheep Alveolar and Mouse Macrophages Infected by Mycoplasma ovipneumoniae [J]. Fujian Journal of Agricultural Sciences,2020,35(11):1244−1251 doi: 10.19303/j.issn.1008-0384.2020.11.010
Citation: ZHANG Y, ZHANG K, MA J, et al. Proteomes of Sheep Alveolar and Mouse Macrophages Infected by Mycoplasma ovipneumoniae [J]. Fujian Journal of Agricultural Sciences,2020,35(11):1244−1251 doi: 10.19303/j.issn.1008-0384.2020.11.010

绵羊肺炎支原体感染对绵羊肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7蛋白质组的影响

doi: 10.19303/j.issn.1008-0384.2020.11.010
基金项目: 国家自然科学基金项目(31960036、31460039);教育部“春晖计划”国际合作项目(NXU201802)
详细信息
    作者简介:

    张颖(1996−),女,硕士研究生,研究方向:细胞生物学(E-mail:1776244258@qq.com

    通讯作者:

    高力扬(1981−),女,博士,讲师,研究方向:细胞生物学(E-mail:pandarun@nxu.edu.cn

  • 中图分类号: Q 233

Proteomes of Sheep Alveolar and Mouse Macrophages Infected by Mycoplasma ovipneumoniae

  • 摘要:   目的  评估小鼠巨噬细胞系Raw 264.7替代绵羊肺泡巨噬细胞用于研究绵羊肺炎支原体(Mycoplasma ovipneumoniae, Mo)致病机制的可行性。  方法  从绵羊肺脏灌洗液中分离绵羊原代肺泡巨噬细胞,以绵羊原代肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7细胞系为细胞模型,使用绵羊肺炎支原体Y98标准株分别感染(MOI=10)绵羊原代肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7细胞系24 h后,通过蛋白质组学或定时荧光定量PCR检测Mo刺激后两种细胞中部分蛋白或基因的相对表达量。  结果  从肺中成功分离出绵羊原代肺泡巨噬细胞,经免疫荧光鉴定显示其带有巨噬细胞特异性表面抗原CD14;经Mo感染后,绵羊原代肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7中FADDIL-1βNOS2THBS1等基因的表达均发生显著变化,主要涉及Toll样受体信号通路、MAPK信号通路、自噬作用等生物过程且两种细胞各基因的相对表达变化趋势基本一致。  结论  本研究初步表明选用小鼠巨噬细胞Raw 264.7细胞系替代绵羊原代巨噬细胞进行与Mo的互作研究具有一定的可行性,可为简化Mo致病机制研究模型提供理论基础。
  • 图  1  绵羊原代肺泡巨噬细胞的鉴定

    注:A. 绵羊原代肺泡巨噬细胞形态学观察;B. 绵羊原代肺泡巨噬细胞的CD14免疫荧光鉴定(CD14:巨噬细胞特异性表面抗原;DAPI:细胞核;Merge: CD14与DAPI的重叠图)。

    Figure  1.  Identification of isolated sheep primary alveolar macrophage

    Note: A: Morphology of sheep primary alveolar macrophages; B: Immunofluorescence identification of sheep primary alveolar macrophage by CD14 antibody (CD14: Macrophage specific surface antigen; DAPI: Nucleus; Merge: Overlap of CD14 and DAPI).

    图  2  Mo进入绵羊肺泡巨噬细胞和小鼠巨噬细胞Raw 264.7后的定位

    注:A. Mo在绵羊肺泡巨噬细胞内的定位;B. Mo在小鼠巨噬细胞Raw 264.7内的定位。

    Figure  2.  Mo localization after entering sheep alveolar macrophage and Raw 264.7

    Note: A: Mo localization in sheep alveolar macrophage; B: Mo localization in Raw 264.7.

    图  3  绵羊肺泡巨噬细胞感染Mo后相关分子的表达变化

    注:A:绵羊原代肺泡巨噬细胞感染Mo后的蛋白聚类分析;B:Toll样受体信号通路中相关蛋白的表达差异;C:MAPK信号通路中相关蛋白的表达;D:ECM受体相互作用中相关蛋白的表达差异;E:自噬作用中相关蛋白的表达差异。***、**、*分别表示处理间在0.001、0.01、0.05水平差异显著。

    Figure  3.  Changes on expressions of target proteins in sheep alveolar macrophage infected by Mo

    Note: A: Protein clusters in sheep alveolar macrophage infected by Mo; B: Expression change on protein related to Toll-like receptor signal pathway; C: Expression change on protein related to MAPK signal pathway; D: Expression change on protein related to ECM receptor interaction; E: Expression change on protein related to autophagy. *** P<0.001 vs. control; **0.001<P<0.01 vs. control; *0.01<P<0.05 vs. control.

    图  4  小鼠巨噬细胞Raw 264.7感染Mo后相关分子的表达变化

    注:A:Toll样受体信号通路中相关基因mRNA的表达差异;B:MAPK信号通路中相关基因mRNA的表达差异;C:ECM受体相互作用中相关基因mRNA的表达差异;D:自噬作用中相关基因mRNA的表达差异。***、**、*分别表示处理间在0.001、0.01、0.05水平差异显著。

    Figure  4.  Changes on expressions of target proteins in Raw 264.7 infected by Mo

    Note: A: Expression change on mRNA related to Toll-like receptor signal pathway; B: Expression change on mRNA related to MAPK signal pathway; C: Expression change on mRNA related to ECM receptor interaction; D: Expression change on mRNA related to autophagy. *** P <0.001 vs. control; **0.001<P <0.01 vs. control; * 0.01<P <0.05 vs. control.

    图  5  绵羊肺泡巨噬细胞和小鼠巨噬细胞感染Mo后相关分子表达变化的对比

    注:A. 绵羊肺泡巨噬细胞感染Mo后相关分子的表达变化 B.小鼠巨噬细胞Raw 264.7感染Mo后相关分子的表达变化。橙色代表上调表达的基因,绿色代表下调表达的基因。

    Figure  5.  Expression changes on proteins in Mo-infected sheep alveolar macrophage vs. Mo-infected Raw 264.7

    Note:A: Expression changes on proteins in sheep alveolar macrophage infected by Mo; B: Expression changes on proteins in Raw 264.7 infected by Mo. Orange color indicates upregulated genes, and green color downregulated genes.

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出版历程
  • 收稿日期:  2020-06-12
  • 修回日期:  2020-09-04
  • 网络出版日期:  2020-11-24
  • 刊出日期:  2020-11-30

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