TAK1调控奶牛乳腺上皮细胞炎症因子和趋化因子的机制  

Mechanism of TAK1 in Regulating Inflammatory Factors and Chemokines in Cow Mammary Epithelial Cells

作　　者：陈平 王斐[1] 谢建鹏 张鉴 何振富[1] CHEN Ping;WANG Fei;XIE Jianpeng;ZHANG Jian;HE Zhenfu(Animal Husbandry and Green Agricultural Institute,Gansu Academy of Agricultural Sciences,Lanzhou 730070,China)

机构地区：[1]甘肃省农业科学院畜草与绿色农业研究所,甘肃兰州730070

出　　处：《中国兽医杂志》2025年第2期18-24,共7页Chinese Journal of Veterinary Medicine

基　　金：甘肃省科协2023年创新驱动助力工程项目(GXH20230817-9);甘肃省农业科学院农业科技自主创新专项博士基金项目(2021GAAS51)。

摘　　要：为了探索转化生长因子β激活激酶1(TAK1)调控奶牛乳腺上皮细胞(CMECs)炎症因子和趋化因子的机制,本试验通过生物信息学分析正常和乳腺炎CMECs信号通路;CMECs经细胞角蛋白18(CK-18)免疫荧光染色鉴定后分为对照组、过表达组和抑制组,对照组正常培养CMECs,过表达组CMECs转染2μg TAK1过表达质粒,抑制组CMECs转染2μg TAK1过表达质粒后加入TAK1抑制剂5Z-7-oxozeaenol,蛋白质免疫印迹(WB)检测CMECs中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、核转录因子κB(NF-κB)、单核细胞趋化蛋白-1(MCP-1)、巨噬细胞炎性蛋白1α(MIP-1α)和巨噬细胞炎性蛋白1β(MIP-1β)含量,流式细胞仪检测CMECs干细胞标志物乙醛脱氢酶(ALDH)含量。生物信息学分析结果显示,CMECs发生炎症时,细胞免疫应答和细胞表面受体信号通路被激活,参与正向调控炎症反应的相关基因呈高水平表达;TNF-α与IL-1β、MIP-1α、MIP-1β、MCP-1、上游蛋白分化簇3(CD3)、分化簇8(CD8)和趋化因子配体5(CCL5)之间存在相互作用。WB结果显示,与对照组相比,过表达组TAK1,炎症因子IL-1β、TNF-α和NF-κB,趋化因子MIP-1α、MIP-1β和MCP-1蛋白表达极显著增强(P<0.01或P<0.001);与过表达组相比,抑制组TAK1,炎症因子IL-1β、TNF-α和NF-κB,趋化因子MIP-1α、MIP-1β和MCP-1蛋白表达极显著受到抑制(P<0.001)。流式细胞仪检测发现,培养48 h之后,对照组ALDH阳性细胞率为6.7%,过表达组ALDH阳性细胞率为10.21%,抑制组ALDH阳性细胞率降为4.23%。本试验探究了TAK1与CMECs炎症因子和趋化因子之间的调控关系,为进一步治疗奶牛乳腺炎提供一定的试验支撑和理论依据。To explore the regulatory mechanism of transforming growth factorβactivated kinase 1(TAK1)on inflammatory factors and chemokines in cow mammary epithelial cells(CMECs),this study performed a bioinformatics analysis of the signaling pathways in normal and mastitis-affected CMECs.CMECs were identified via cytokeratin 18(CK-18)immunofluorescence staining and divided into three groups:Control group,Overexpression group,and Inhibition group.The Control group involved normal CMEC culture,the Overexpression group involved CMECs transfected with 2μg TAK1 overexpression plasmid,and the Inhibition group involved CMECs transfected with 2μg TAK1 overexpression plasmid followed by treatment with the TAK1 inhibitor 5Z-7-oxozeaenol.Western blot(WB)was used to detect levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),nuclear factor-κB(NF-κB),monocyte chemoattractant protein-1(MCP-1),macrophage inflammatory protein-1α(MIP-1α),and macrophage inflammatory protein-1β(MIP-1β).Aldehyde dehydrogenase(ALDH),a CMEC stem cell marker,was measured via flow cytometry.Bioinformatics analysis revealed activation of cellular immune responses and cell surface receptor signaling pathways in inflamed CMECs,with high-level expression of genes positively regulating inflammation.TNF-αinteracted with IL-1β,MIP-1α,MIP-1β,MCP-1,upstream proteins cluster of differentiation 3(CD3)and cluster of differentiation 8(CD8),and chemokine ligand 5(CCL5).WB results showed that compared to the Control group,the Overexpression group exhibited significantly elevated levels of TAK1,inflammatory factors IL-1β,TNF-αand NF-κB,chemokines MIP-1α,MIP-1β,and MCP-1 proteins(P<0.01 or P<0.001),while these protein levels were significantly suppressed in the Inhibition group compared to the Overexpression group(P<0.001).Flow cytometry revealed ALDH-positive cell rates of 6.7%,10.21%,and 4.23%in the Control,Overexpression,and Inhibition groups,respectively,after 48 hours of culture.This study elucidates the regulatory relationship between TAK1 an

关 键 词：奶牛乳腺上皮细胞(CMECs) 转化生长因子β激活激酶1(TAK1) 炎症因子 相互作用 

分 类 号：S852.21[农业科学—基础兽医学]