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

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

1.
宁夏大学生命科学学院/西部特色生物资源保护与利用教育部重点实验室，宁夏　银川　750021
2.
宁夏大学新华学院，宁夏　银川　750021

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
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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

Proteomes of Sheep Alveolar and Mouse Macrophages Infected by Mycoplasma ovipneumoniae

1.
School of Science, Ningxia University/Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in Western China
2.
Xinhua College, Ningxia University, Yinchuan, Ningxia　750021, China

摘要

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FADD、IL-1β、NOS2及THBS1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。
- 绵羊肺炎支原体 /
- 绵羊肺泡巨噬细胞 /
- Raw 264.7细胞 /
- 细胞模型
Abstract: Objective Feasibility of using mouse macrophage cell line Raw 264.7 for studies on pathogenesis of Mycoplasma ovipneumoniae(Mo) on sheep was investigated by comparing the proteome changes taken place in both macrophages after an induced Mo infection. Method The primary alveolar macrophages from sheep lung tissue were isolated and used, along with Raw 264.7, for the study. Both macrophages were infected with a standard strain of Mo (MOI=10) for 24h prior to proteomic and RT-PCR analyses to compare the expressions on the target genes in them. Result The image of specific surface antigen CD14 shown on the cells obtained from the lung tissue confirmed the identity of the isolated sheep primary alveolar macrophages. After infected by Mo, both sheep and mouse macrophages showed a similar significant change pattern on the expressions of FADD, IL-1β, NOS2, and THBS1. These proteins are known to associated with the Toll-like receptor signaling pathway, MAPK signaling pathway, and autophagy in biological processes. Conclusion It appeared that Raw 264.7 could be satisfactorily used to substitute the sheep primary alveolar macrophage in a simulated system for studies on Mo pathogenesis to considerably simplify the process and save cost in conducting the experiments on sheep.
- Mycoplasma ovipneumoniae /
- sheep alveolar macrophages /
- Raw 264.7 /
- cell model

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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).

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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.

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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.

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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.

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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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