脂磷壁酸体外诱导奶牛乳腺上皮细胞炎症模型的建立与评价
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摘要
旨在通过脂磷壁酸(LTA)诱导的方法,体外建立奶牛乳腺上皮细胞(BMECs)炎症模型,为奶牛乳腺炎的研究提供合理模型。采用Ⅱ型胶原酶消化法分离培养BMECs,利用差时胰酶消化法纯化BMECs。通过测定BMECs骨架蛋白——角蛋白18对所培养的细胞进行鉴定,以确定其为BMECs,并用LTA侵染BMECs,以培养细胞的形态学特征和炎性因子作为炎症发生和发展的判断指标。用不同质量浓度(0、10、20、40、80 g/m L)的LTA对BMECs作用不同时间(12、24、48 h),采用MTT法检测细胞活力;采用酶联免疫吸附试验(ELISA)快速检测奶牛肿瘤坏死因子(TNF-α)和奶牛白细胞介素1β(IL-1β)的蛋白表达水平;采用实时荧光定量PCR(qRT-PCR)检测炎性应答标志TNF-α和IL-1β基因的表达水平,以确定建立模型的最佳质量浓度与时间。结果表明,在体外培养的BMECs特异性蛋白——角蛋白18表达呈阳性;MTT、ELISA和qRT-PCR试验结果显示LTA质量浓度为40 g/m L时处理BMECs 24 h可明显提高TNF-α和IL-1β的蛋白和基因表达水平。
To establish an inflammation model of bovine mammary epithelial cells(BMECs)in vitro by lipoteichoic acid(LTA)-induced method,and to provide a reasonable model for the study of bovine mastitis,BMECs were isolated and cultured by type Ⅱcollagenase digestion method, and purified by differential trypsin digestion.The cultured cells were identified by measuring bovine mammary epithelial-derived keratin 18 and infected with LTA.Morphological features and inflammatory factors of the cultured cells were used as indicators of the occurrence and development of inflammation.The effects of LTA at different concentrations(0,10,20,40,and 80 g/m L) on BMECs were observed at different time points(12,24 and 48 h).The MTT method was used to determine BMECs activity.The enzyme-linked immunosorbent assay(ELISA) was used to detect the protein expression levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β).Real-time quantitative PCR(qRT-PCR) was used to detect the expression of TNF-α and IL-1β in inflammatory response markers to determine the optimal concentration and time for establishing the model.The results showed that the expression of keratin 18,a BMECs specific protein,was positive in vitro;MTT,ELISA and qRT-PCR showed that the treatment of BMECs with LTA at 40 g/m L for 24 h obviously improved TNF-α and IL-1β protein and gene expression levels.
To establish an inflammation model of bovine mammary epithelial cells(BMECs)in vitro by lipoteichoic acid(LTA)-induced method,and to provide a reasonable model for the study of bovine mastitis,BMECs were isolated and cultured by type Ⅱcollagenase digestion method, and purified by differential trypsin digestion.The cultured cells were identified by measuring bovine mammary epithelial-derived keratin 18 and infected with LTA.Morphological features and inflammatory factors of the cultured cells were used as indicators of the occurrence and development of inflammation.The effects of LTA at different concentrations(0,10,20,40,and 80 g/m L) on BMECs were observed at different time points(12,24 and 48 h).The MTT method was used to determine BMECs activity.The enzyme-linked immunosorbent assay(ELISA) was used to detect the protein expression levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β).Real-time quantitative PCR(qRT-PCR) was used to detect the expression of TNF-α and IL-1β in inflammatory response markers to determine the optimal concentration and time for establishing the model.The results showed that the expression of keratin 18,a BMECs specific protein,was positive in vitro;MTT,ELISA and qRT-PCR showed that the treatment of BMECs with LTA at 40 g/m L for 24 h obviously improved TNF-α and IL-1β protein and gene expression levels.
引文
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[2]EBNER K E,HOOVER C R,HAGEMAN E C,et al.Cultivation and properties of bovine mammary cell cultures[J].Experimental Cell Research,1961,23(2):373.
[3]PARK H J,LEE W Y,JEONG H Y,et al.Regeneration of bovine mammary gland in immunodeficient mice by transplantation of bovine mammary epithelial cells mixed with matrigel[J].International Journal of Stem Cells,2016,9(2):186-191.
[4]BOUGARN S,CUNHA P,HARMACHE A,et al.Muramyl dipeptide synergizes with Staphylococcus aureus lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells[J].Clinical and Vaccine Immunology,2010,17(11):1797.
[5]刘莉莉.脂肪酸与PPARγ基因互作对奶牛乳腺上皮细胞乳脂肪合成的调节作用及机理[D].哈尔滨:东北农业大学,2014.LIU Li-li.The effect of interaction between fatty acid and PPARγgene on regulation and mechanism of milk fat synthesis in dairy cow mammary epithelial cells[D].Harbin:Northeast Agricultural University,2014.(in Chinese)
[6]董海龙,吴琼,刘明超,等.奶牛乳腺上皮细胞的体外分离培养及超微结构鉴定[J].中国兽医学报,2016,36(10):1763-1768.DONG Hai-long,WU Qiong,LIU Ming-chao,et al.Inprimary culture and ultrastructural changes of bovine mammary epithelial cells[J].Chinese Journal of Veterinary Science,2016,36(10):1763-1768.(in Chinese)
[7]陈秋菊,王韵斐,崔易虹,等.组织块再移法分离培养奶山羊乳腺上皮细胞[J].中国兽医学报,2012,32(2):301-304.CHEN Qiu-ju,WANG Yun-fei,CUI Yi-hong,et al.Study on tissue re-shift method of isolation and culture of goat mammary epithelial cells[J].Chinese Journal of Veterinary Science,2012,32(2):301-304.(in Chinese)
[8]朝格图.奶牛乳腺上皮细胞和成纤维细胞原代培养与鉴定[D].呼和浩特:内蒙古大学,2015.CHAO Ge-tu.Primary culture and identification of bovine mammary epithelial cell and fibroblast cell[D].Hohhot:Inner Mongolia University,2015.(in Chinese)
[9]JACK H T,UMNIYATI S R,MURHANDARWATI E H.Comparison of immunochromatography method and immunocytochemistry method in rapid detection of NS-1antigen in dengue infection[J].Tropical Medicine Journal,2013,3(1):48-56.
[10]WU T,WANG C,DING L,et al.Arginine relieves the inflammatory response and enhances the casein expression in bovine mammary epithelial cells induced by lipopolysaccharide[J].Mediators of Inflammation,2016,2016(4):1-10.
[11]马驿,孙永学,陈进军,等.兽药残留对生态环境影响的研究进展[J].中国兽医科学,2010,40(6):650-654.MA Yi,SUN Yong-xue,CHEN Jin-jun,et al.Research progress on the impact of veterinary drug residues on the ecological environment[J].Chinese Veterinary Science,2010,40(6):650-654.(in Chinese)
[12]林杰.植物精油对LPS诱导的奶牛乳腺上皮细胞损伤的保护作用研究[D].北京:中国农业科学院研究生院,2016.LIN Jie.Study of plant-derived essential oil protection injury of bovine mammary epithelial cells induced by lipopolysaccharide[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[13]刘建洲.基于BEAS-2B细胞模型的青翘提取物抗炎活性研究[D].太原:山西大学,2015.LIU Jian-zhou.Anti-inflammatory activity of Forsythia Suspensa extract on human airway epithelial cells inflammation model[D].Taiyuan:Shanxi University,2015.(in Chinese)
[14]况玲,历世伟,武聚富,等.成纤维上皮细胞炎症反应模型的建立[J].中国兽医学报,2011,31(7):1054-1058.KUANG Ling,LI Shi-wei,WU Ju-fu,et al.Establishment of inflammatory response models in fibroid epithelial cell[J].Chinese Journal of Veterinary Science,2011,31(7):1054-1058.(in Chinese)
[15]GIOVANNINI A E J,WALL S K,WELLNITZ O,et al.Experimentally induced subclinical mastitis:Are lipopolysaccharide and lipoteichoic acid eliciting similar pain responses[J].Acta Veterinaria Scandinavica,2017,59(1):40.
[16]崔新洁,胡庆亮,李奕平,等.金黄色葡萄球菌诱导牛原代乳腺上皮细胞的凋亡[J].中国农业科学,2013,46(15):3212-3219.CUI Xin-jie,HU Qing-liang,LI Yi-ping,et al.The apoptosis of bovine primary mammary epithelial cells induced by Staphylococcus aureus[J].Scientia Agriculture Sinica,2013,46(15):3212-3219.(in Chinese)
[17]陈亚冰,符梅,兰道亮,等.牦牛乳腺上皮细胞体外培养及鉴定[J].畜牧兽医学报,2015,46(1):69-76.CHEN Ya-bing,FU Mei,LAN Dao-liang,et al.Culture and identification of yak mammary epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2015,46(1):69-76.(in Chinese)
[18]黄国珍,陈伟布,李丽梅,等.双歧杆菌的脂磷壁酸激活巨噬细胞产生TNF-α的信号通路[J].中国当代医药,2013,20(21):102-103.HUANG Guo-zhen,CHEN Wei-bu,LI Li-mei,et al.Lipoteichoic acid of Bifidobacterium activate macrophages to produce TNF-αsignaling pathway[J].China Modern Medicine,2013,20(21):102-103.(in Chinese)
[19]林晶晶,陶慧,侯瑛倩,等.脂磷壁酸对小鼠子宫内膜组织和内膜细胞中功能受体TLR2表达的影响[J].畜牧兽医学报,2013,44(5):811-816.LIN Jing-jing,TAO Hui,HOU Ying-qian,et al.Effect of lipoteichoic acid on functional receptor TLR2 expression in endometrial tissue and cell of mice[J].Acta Veterinaria et Zootechnica Sinica,2013,44(5):811-816.(in Chinese)