NFκB1在奶牛乳腺上皮细胞中的表达与定位  

Expression and Localization of NFκB1 in Bovine Mammary Epithelial Cell

作　　者：臧艳丽[1] 古新宇 黄鑫[1] 于翠平[1] 骆超超[1] 高学军[1] 

机构地区：[1]东北农业大学,黑龙江省高校农业生物功能基因重点实验室,哈尔滨150030

出　　处：《中国畜牧兽医》2016年第8期2112-2119,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家高技术研究发展计划(863计划)项目(2013AA102504-03)

摘　　要：试验旨在研究奶牛乳腺上皮细胞(bovine mammary epithelial cell,BMEC)中NFκB1的表达与定位变化,以阐明NFκB1对BMEC乳合成的转录调节机理。通过组织块法培养原代细胞,利用BMEC和成纤维细胞对胰蛋白酶敏感性的不同进行纯化和传代;利用Western blotting和免疫荧光染色法检测细胞中角蛋白18和β-酪蛋白的表达以鉴定细胞的纯度和泌乳功能;通过添加0.6mmol/L蛋氨酸建立氨基酸刺激BMEC泌乳的体外模型;Western blotting和免疫荧光法检测添加蛋氨酸后NFκB1和p-NFκB1表达与定位的变化。结果显示,获得了纯化的BMEC;Western blotting检测发现NFκB1在细胞浆中存在约141和105ku两种形式,在细胞核中只存在105ku形式;p-NFκB1(50ku)主要存在于细胞核内。在添加蛋氨酸后NFκB1的141ku形式的含量有一定增加;105ku形式在细胞浆内蛋白水平变化不明显,在细胞核内水平明显升高;p-NFκB1在细胞核中的定位明显增加。结果提示,NFκB1参与BMEC乳合成的转录调节,氨基酸通过促进细胞核内NFκB1磷酸化以调节泌乳相关基因的转录和促进乳合成。The experiment was aimed to study the expression and localization of NFκB1in bovine mammary epithelial cell（BMEC）,and to clarify the regulation of transcription for milk synthesis in BMEC by NFκB1.Primary cells were cultured using the tissue pieces culture method,and the BMEC was purified and passaged according to the different sensitivity of fibroblasts and BMEC to trypsin.Western blotting and immunofluorescence（IF）method were used to detect cell keratin 18andβ-casein expression to identify the cell purity and lactogenesis function.Furthermore,0.6mmol/L methionine（Met）was added to BMEC culture to establish amino acids-stimulating BMEC model in vitro.Western blotting and IF were used to detect the expression and localization of NFκB1and p-NFκB1after adding Met.These results showed that BMEC was successfully purified.NFκB1had two molecular forms in cytoplasm,the molecular weights were about 141and105 ku,and in nucleus only 105 ku form exists.The p-NFκB1（50ku）mainly existed in the nucleus.After Met stimulation,the protein level of NFκB1 141 ku form was slightly upregulated,the protein level of NFκB1 105 ku did not changed in the cytoplasm while obviously increased in the nucleus,the protein level and nuclear localization of p-NFκB1 were both obviously increased.These results suggested that NFκB1was involved in the transcriptional regulation of lactogenesis in BMEC,and amino acids promoted lactogenesis by triggering NFκB1phosphorylation in the nucleus.

关 键 词：NFκB1 p-NFκB1 奶牛乳腺上皮细胞 蛋氨酸 乳合成 

分 类 号：S814.8[农业科学—畜牧学]