NFκB1在奶牛乳腺上皮细胞中的表达与定位

doi:10.16431/j.cnki.1671-7236.2016.08.025

摘要/Abstract

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0.6 mmol/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105 ku两种形式，在细胞核中只存在105 ku形式；p-NFκB1（50 ku）主要存在于细胞核内。在添加蛋氨酸后NFκB1的141 ku形式的含量有一定增加；105 ku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。

关键词: NFκ B1; p-NFκ B1; 奶牛乳腺上皮细胞; 蛋氨酸; 乳合成

Abstract:

The experiment was aimed to study the expression and localization of NFκB1 in 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 18 and β-casein expression to identify the cell purity and lactogenesis function.Furthermore,0.6 mmol/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 NFκB1 and p-NFκB1 after adding Met.These results showed that BMEC was successfully purified.NFκB1 had two molecular forms in cytoplasm,the molecular weights were about 141 and 105 ku,and in nucleus only 105 ku form exists.The p-NFκB1 (50 ku) 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 NFκB1 was involved in the transcriptional regulation of lactogenesis in BMEC,and amino acids promoted lactogenesis by triggering NFκB1 phosphorylation in the nucleus.

Key words: NFκB1; p-NFκB1; BMEC; methionine; lactogenesis

中图分类号:

S814.8

臧艳丽, 古新宇, 黄鑫, 于翠平, 骆超超, 高学军. NFκB1在奶牛乳腺上皮细胞中的表达与定位[J]. 《中国畜牧兽医》, 2016, 43(8): 2112-2119.

ZANG Yan-li, GU Xin-yu, HUANG Xin, YU Cui-ping, LUO Chao-chao, GAO Xue-jun. Expression and Localization of NFκB1 in Bovine Mammary Epithelial Cell[J]. , 2016, 43(8): 2112-2119.

参考文献

[1] Chen F,Castranova V,Shi X,et al.New insights into the role of nuclear factor-kappaB,a ubiquitous transcription factor in the initiation of diseases[J].Clin Chem,1999,45(1):7-17.
[2] Collins P E,Kiely P A,Carmody R J.Inhibition of transcription by B cell Leukemia 3(Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50[J].J Biol Chem,2014,289(10):7059-7067.
[3] Li H P,Tao Y Q,Zhao P,et al.Effects of Pholiota nameko polysaccharide on NF-κB pathway of murine bone marrow-derived dendritic cells[J].Int J Biol Macromol,2015,77:120-130.
[4] Lv Y N,Li S X,Zhai K F,et al.Network pharmacology-based prediction and verification of the molecular targets and pathways for schisandrin against cerebrovascular disease[J]. Chin J Nat Med,2014,12(4):251-258.
[5] Nair J,Ghatge M,Kakkar V V,et al.Network analysis of inflammatory genes and their transcriptional regulators in coronary artery disease[J].PLoS One,2014,9(4):e94328.
[6] Yang Y N,Zhang J Y,Ma Y T,et al.-94 ATTG insertion/deletion polymorphism of the NFκB1 gene is associated with coronary artery disease in Han and Uygur women in China[J].Genet Test Mol Bioma,2014,18(6):430-438.
[7] Burkitt M D,Williams J M,Duckworth C A,et al.Signaling mediated by the NF-κB sub-units NF-κB1,NF-κB2 and c-Rel differentially regulate Helicobacter felis-induced gastric carcinogenesis in C57BL/6 mice[J].Oncogene,2013,32(50):5563-5573.
[8] Richert M M,Schwertfeger K L,Ryder J W,et al.An atlas of mouse mammary gland development[J].J Mammary Gland Biol,2000,5(2):227-241.
[9] Sternlicht M D.Key stages in mammary gland development:The cues that regulate ductal branching morphogenesis[J]. Breast Cancer Res Bcr,2006,8(1):201.
[10] Clarke C,Sandle J,Lakhani S R.Myoepithelial cells:Pathology,cell separation and markers of myoepithelial differentiation[J].J Mammary Gland Biol,2005,10(3):273-280.
[11] 王杰,刁其玉,张乃锋.微量营养素与脂肪对动物基因表达的调控作用[J].中国畜牧兽医,2016,43(1):140-146.
[12] Horseman N D.Mammary gland development[J].Advances in Dental Research,1995,1(4):533-557.
[13] Pawlowski K M,Popielarz D,Szyszko K,et al.Growth hormone receptor (GHR) RNAi decreases proliferation and enhances apoptosis in CMT-U27 canine mammary carcinoma cell line[J].Vet Comp Oncol,2012,10(1):2-15.
[14] Song Y,Santen R J,Wang J P,et al.Effects of the conjugated equine estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) on mammary gland and breast cancer in mice[J].Endocrinology,2012,153(12):5706-5715.
[15] Shushanov S S.Insulin-like growth factors 1and 2 regulate expression of β-casein in vitro in mouse mammary epithelial cells[J].B Exp Biol Med,2011,152(2):202-205.
[16] Wiesen J,Young P Z,Cunha G.Signaling through the stromal epidermal growth factor receptor is necessary for mammary ductal development[J].Development,1999,126(2):335-344.
[17] Sinowatz F,Schams D,Habermann F,et al.Localization of fibroblast growth factor Ⅰ (acid fibroblast growth factor) and its mRNA in the bovine mammary gland during mammogenesis,lactation and involution[J].Anant Histol Embryol,2006,35(3):202-207.
[18] Rius A G,Appuhamy J,Cyriac J,et al.Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids[J].J Dairy Sci,2010,93(7):3114-3127.
[19] Burgos S A,Dai M,Cant J P.Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway[J].J Dairy Sci,2010,93(1):153-161.
[20] Anderson S M,Rudolph M C,Mcmanaman J L,et al.Secretory activation in the mammary gland:It's not just about milk protein synthesis[J].Breast Cancer Res,2007,9(1):204.
[21] Yuan H X,Xiong Y,Guan K L.Nutrient sensing,metabolism,and cell growth control[J].Mol Cell,2013,49(3):379-387.
[22] 刘怀禹,李浩天,苏小虎,等.Sox2基因真核表达载体的构建及转Sox2基因绵羊骨髓间充质干细胞系的建立[J].中国畜牧兽医,2016,43(1):16-22.
[23] Hayden M S,Ghosh S.Shared principles in NF-kappaB signaling[J].Cell,2008,132(3):344-362.
[24] Bonizzi G,Karin M.The two NF-κB activation pathways and their role in innate and adaptive immunity[J].Trends Immunol,2004,25(6):280-288.
[25] Liang Y,Zhou Y,Shen P.NF-κB and its regulation on the immune system[J].Cell Mol Immunol, 2004,1(5):343-350.
[26] Hellweg C E.The Nuclear Factor κB pathway:A link to the immune system in the radiation response[J].Cancer Lett,2015,368(2):275-289.
[27] Perkins N D.Integrating cell-signalling pathways with NF-kappaB and IKK function[J].Nat Rev Mol Cell Bio,2007,8(1):49-62.