乳腺炎对牛奶外泌体功能的影响：基于乳腺上皮细胞的观察

doi:10.3785/j.issn.1008-9209.2019.07.181

浙江大学学报（农业与生命科学版）

2020, Vol. 46

Issue (3): 383-390 DOI: 10.3785/j.issn.1008-9209.2019.07.181

动物科学与动物医学

乳腺炎对牛奶外泌体功能的影响：基于乳腺上皮细胞的观察

应易恬(

),杨璟,严冰璇,邵冯金,谭勋(

)

浙江大学动物科学学院，杭州 310058

Effect of mastitis on the function of milk-derived exosomes: observations from mammary epithelial cells

Yitian YING(

),Jing YANG,Bingxuan YAN,Fengjin SHAO,Xun TAN(

)

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

全文: PDF(1471 KB) HTML

摘要：

通过观察乳腺炎牛奶外泌体对乳腺上皮细胞（mammary epithelial cells, MECs）活力和天然免疫功能的影响，探究外泌体在乳腺炎发病机制中的作用。MECs经热灭活的乳腺炎病原（大肠埃希菌）刺激24 h后进行转录组测序和基因本体（gene ontology, GO）富集分析，发现有21条差异表达基因富集在细胞外囊泡外泌体（GO: 0070062）这一细胞组分上，提示感染可引起宿主细胞外泌体生物学功能改变。在此基础上，采用正常牛奶来源的外泌体（N-exo）和乳腺炎牛奶来源的外泌体(M-exo)作用MECs，以培养于不含外泌体的培养基中的细胞为对照，采用3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐（methyl-thiazol-diphenyltetrazolium, MTT）法检测细胞活力，并采用实时荧光定量聚合酶链式反应检测MECs中白细胞介素8（interleukin-8, IL-8）、白细胞介素1β（interleukin-1β, IL-1β）和肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）的表达。结果发现：正常牛奶外泌体对MECs活力无显著影响，而乳腺炎牛奶外泌体可显著抑制MECs活力；2种来源的外泌体对IL-8和TNF-α的表达均无显著影响，但正常牛奶外泌体可诱导表达IL-1β，而乳腺炎牛奶外泌体则缺乏诱导IL-1β表达的能力。上述结果提示，在乳腺感染过程中，进入牛奶中的外泌体可引起MECs活力下降，并可能参与介导有利于病原逃避免疫的微环境的形成。因而，在乳腺炎发病机制中，外泌体可能起着促进乳腺感染扩散的作用。

关键词： 牛奶; 外泌体; 乳腺上皮细胞; 促炎细胞因子

Abstract:

The present study was conducted to investigate the effect of mastitic milk-derived exosomes on the viability and innate immune function of bovine mammary epithelial cells (MECs), with the aim to understand the role of exosomes in the pathogenesis of mastitis. Primary MECs were stimulated with heat-killed mastitis causing bacteria (Escherichia coli) for 24 h and subjected to RNA-sequencing and gene ontology (GO) functional classification analysis. It was found that 21 differential expression genes between pathogen-stimulated cells and normal cells were enriched in the cellular component “extracellular vesicular exosome” (GO: 0070062), suggesting that infection may alter the physiological function of exosomes derived from host cells. Based on this finding, MECs were stimulated with the exosomes derived from normal milk (N-exo) and mastitic milk (M-exo), respectively, for 24 h. The cells cultured in the exosome-depleted medium were served as the controls. The cell viability was determined by methyl-thiazol-diphenyltetrazolium (MTT) assay, and the expression of pro-inflammatory cytokines interleukin-8 (IL-8), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the MECs was measured by quantitative real-time PCR (qPCR). The results showed that the exosomes derived from normal milk (N-exo) had no effect on the MEC viability, and those isolated from mastitic milk (M-exo) led to a significant reduction in the cell viability. The exosomes derived from both sources had no effect on the expression of IL-8 and TNF-α. However, the normal milk-derived exosomes significantly induced the expression of IL-1β; in contrast, the exosomes derived from mastitic milk failed to stimulate IL-1β expression. The above results suggest that the exosomes present in milk in the process of udder infection might reduce MEC viability and mediate the formation of a microenvironment favoring the immune escape of pathogens. Thus, it is very likely that exosomes contribute to the pathogenesis of mastitis by helping spread the infection in udder.

Key words: milk exosome mammary epithelial cell pro-inflammatory cytokine

收稿日期: 2019-07-18 出版日期: 2020-07-17

CLC:

S 852.3

基金资助: 国家重点研发计划(2017YFD0502200);浙江省科技厅项目(2013C32036)

通讯作者: 谭勋 E-mail: 21717097@zju.edu.cn;tanxun@zju.edu.cn

作者简介: 应易恬（https://orcid.org/0000-0001-9079-6759），E-mail：21717097@zju.edu.cn

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引用本文:

应易恬,杨璟,严冰璇,邵冯金,谭勋. 乳腺炎对牛奶外泌体功能的影响：基于乳腺上皮细胞的观察[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(3): 383-390.

Yitian YING,Jing YANG,Bingxuan YAN,Fengjin SHAO,Xun TAN. Effect of mastitis on the function of milk-derived exosomes: observations from mammary epithelial cells. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(3): 383-390.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.07.181 或 http://www.zjujournals.com/agr/CN/Y2020/V46/I3/383

表1 qPCR引物信息

图1 大肠埃希菌刺激细胞和对照细胞之间的差异表达基因火山图FDR：校正后的P值；FC：差异倍数。

图2 差异表达基因中最显著富集的GO条目FC：差异倍数；FDR：校正后的P值。

表2 富集于细胞的组分“细胞外囊泡外泌体”（GO: 0070062）的差异表达基因

图3 外泌体表征A.外泌体的透射电子显微镜观察；B.外泌体标志性蛋白的免疫印迹检测。N-exo：正常牛奶外泌体；M-exo：乳腺炎牛奶外泌体。

图4 正常牛奶外泌体（N-exo）和乳腺炎牛奶外泌体（M-exo）对奶牛乳腺上皮细胞活力的影响CK：未加外泌体。***表示在P＜0.001水平差异有极高度统计学意义；n=5。

图5 牛奶外泌体（N-exo和M-exo）对奶牛乳腺上皮细胞IL-1β（A）和IL-8（B）表达的影响CK：未加外泌体。**表示在P＜0.01水平差异有高度统计学意义；n=3。

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