动物营养学报 >

2014 , Vol. 26 >Issue 9: 2607 - 2614

DOI: https://doi.org/10.3969/j.issn.1006-267x.2014.09.021

反刍与草食动物营养 Ruminants and Herbivorous Animal Nutrition

奶牛乳腺上皮细胞的不同培养方法比较及激素和细胞因子对β-酪蛋白mRNA表达的诱导

展开
  • 1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
    2. 河南农业大学生命科学学院, 郑州 450002;
    3. CAAS-ICRAF农用林业与可持续畜牧业联合实验室, 北京 100193;
    4. 东北农业大学, 食品安全与营养协同创新中心, 哈尔滨 150030
李文清（1979-），女，河南潢川人，博士研究生，研究方向为反刍动物营养。E-mail：liwenqing605@163.com

收稿日期: 2014-04-08

  网络出版日期: 2014-09-10

基金资助

十二五科技支撑计划课题（2012BAD12B02-5）；中国农业科学院科技创新工程（ASTIP-IAS07）；动物营养学国家重点实验室自主课题（2004DA125184G1103）

收起

Comparison on Culture Methods of Bovine Mammary Epithelial Cells and Induction of Hormones and Cytokines on β-Casein mRNA Expression

Expand
  • 1. State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. College of Life Science, Henan Agricultural University, Zhengzhou 450002, China;
    3. CAAS-ICRAF Joint Laboratory on Agroforestry and Sustainable Animal Husbandry, Beijing 100193, China;
    4. Synergetic Innovation Center of Safety and Nutrition, Northeast Agricultural University, Harbin 150030, China

Received date: 2014-04-08

  Online published: 2014-09-10

Fold

摘要

本试验旨在建立一种高效的奶牛乳腺上皮细胞培养方法，并研究不同激素和细胞因子对其表达β-酪蛋白mRNA的诱导作用。分别采用组织块培养法和机械破碎法培养奶牛乳腺上皮细胞，利用成纤维细胞和乳腺上皮细胞对胰酶的敏感性不同，对获得的细胞进行纯化。通过细胞计数法测定纯化细胞的生长曲线，通过免疫荧光组织化学染色法检测角蛋白18的表达，通过实时定量PCR法检测8种不同激素和细胞因子组合培养液诱导细胞表达β-酪蛋白mRNA的效果。结果表明：通过机械破碎法可以分离得到增殖旺盛的奶牛乳腺上皮细胞，与组织块培养法相比，具有细胞迁出速度快等优点。细胞生长曲线呈典型的“S”型。在纯化的乳腺上皮细胞及第20代乳腺上皮细胞中都检测到角蛋白18的表达。100 ng/mL类胰岛素生长因子Ⅰ（IGF-Ⅰ）显著提高了乳腺上皮细胞中β-酪蛋白mRNA的表达（P<0.05）。由结果可知，本试验采用的机械破碎法是一种高效的奶牛乳腺上皮细胞培养方法，100 ng/mL IGF-Ⅰ对细胞中β-酪蛋白mRNA表达的诱导效果最好。

关键词： 原代细胞培养; 乳腺上皮细胞; 激素; &beta; -酪蛋白mRNA

本文引用格式

李文清, 王加启, 南雪梅, 卜登攀 . 奶牛乳腺上皮细胞的不同培养方法比较及激素和细胞因子对β-酪蛋白mRNA表达的诱导[J]. 动物营养学报, 2014 , 26(9) : 2607 -2614 . DOI: 10.3969/j.issn.1006-267x.2014.09.021

Abstract

This experiment was conducted to establish an efficient culture method for bovine mammary epithelial cells, and to research the induction effect of hormones and cytokines on β-casein (CNS2) mRNA expression. Bovine mammary epithelial cells were cultured by tissue mass culture method and mechanical disruption method, respectively. Cells were purified according to different sensitivity to trypsin of fibroblasts and bovine mammary epithelial cells. The growth curve of purified cells was detected by cell counting assay method. The expression of keratin 18 was detected by immunofluorescent histochemistry staining method. CNS2 mRNA expression induced by 8 kinds of hormones and cytokines combination mediums was measured by real time quantified PCR (RT-qPCR). The results showed that vigorous bovine mammary epithelial cells could be obtained by mechanical disruption method. Cell migration speed of mechanical disruption method was faster than that of tissue mass culture method. The growth curve showed a typical ‘S’ type. The expression of cytokeratin 18 was found in purified cells and the 20th generation of cells. The supplementation of 100 ng/mL insulin-like growth factor Ⅰ (IGF-Ⅰ) significantly improved CNS2 mRNA expression in mammary epithelial cells (P<0.05). It is concluded that the mechanical disruption method used in the present experiment is an efficient method for bovine mammary epithelial cells, and 100 ng/mL IGF-Ⅰ shows the best induction effect on CNS2 mRNA expression.

Key words： primary cell culture; mammary epithelial cell; hormone; CNS2 mRNA

参考文献

[1] LIU H Y,ZHAO K,LIU J X.Effects of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in bovine mammary epithelial cells[J].PLoS One,2013,8(6):e66092.

[2] MONZANI P S,BRESSAN F F,MESQUITA L G,et al.β-casein gene expression by in vitro cultured bovine mammary epithelial cells derived from developing mammary glands[J].Genetics and Molecular Research,2011,10(2):604-614.  

[3] SPERONI L,WHITT G S,XYLAS J,et al.Hormonal regulation of epithelial organization in a three-dimensional breast tissue culture model[J].Tissue Engineering Part C:Methods,2014,20(1):42-51.  

[4] SCHMID E,FRANKE W W,GRUND C,et al.An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays[J].Experimental Cell Research,1983,146(2):309-328.  

[5] HUYNH H T,ROBITAILLE G,TURNER J D.Establishment of bovine mammary epithelial cells (MAC-T):an in vitro model for bovine lactation[J].Experimental Cell Research,1991,197(2):191-199.  

[6] GIBSON C A,VEGA J R,BAUMRUCKER C R,et al.Establishment and characterization of bovine mammary epithelial cell lines[J].In Vitro Cellular & Developmental Biology:Animal,1991,27(7):585-594.  

[7] ZAVIZION B,VAN DUFFELEN M,SCHAEFFER W,et al.Establishment and characterization of a bovine mammary myoepithelial cell line[J].In Vitro Cellular & Developmental Biology:-Animal,1996,32(3):149-158.  

[8] GERMAN T,BARASH I.Characterization of an epithelial cell line from bovine mammary gland[J].In Vitro Cellular & Developmental Biology:Animal,2002,38(5):282-292.  

[9] 王加启,反刍动物营养学研究方法[M].北京:现在教育出版社,2011:225-226.

[10] GROVES T D D,LARSON B L.Preparation of specifically labeled milk proteins using bovine mammary-cell cultures[J].Biochimica Et Biophysica Acta:General Subjects,1965,104(2):462-469.  

[11] CLOSE M J,HOWLETT A R,ROSKELLEY C D,et al.Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton[J].Journal of Cell Science,1997,110(22):2861-2871.

[12] KATZ E,STREULI C H.The extracellular matrix as an adhesion checkpoint for mammary epithelial function[J].International Journal of Biochemistry & Cell Biology,2007,39(4):715-726.  

[13] PAYE J M,AKERS R M,HUCKLE W R,et al.Autocrine production of insulin-like growth factor-I (IGF-Ⅰ) affects paracellular transport across epithelial cells in vitro[J].Cell Communication and Adhesion,2007,14(2/3):85-98.

[14] GRIESBECK-ZILCH B,MEYER H H D,KUHN C H,et al.Staphylococcus aureus and Escherichia coli cause deviating expression profiles of cytokines and lactoferrin messenger ribonucleic acid in mammary epithelial cells[J].Journal of Dairy Science,2008,91(6):2215-2224.  

[15] HU H,WANG J Q,BU D,et al.In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow[J].PLoS One,2009,4(11):e7636.

[16] 弓超,王凌云,周欢敏.牛乳腺上皮细胞体外分离和培养[J].动物医学进展,2011,32(7):66-71.

[17] NOVARO V,ROSKELLEY C D,BISSELL M J.Collagen-Ⅳ and laminin-1 regulate estrogen receptor αexpression and function in mouse mammary epithelial cells[J].Journal of Cell Science,2003,116(14):2975-2986.  

[18] 佟慧丽,尹德云,李庆章,等.奶山羊乳腺上皮细胞系的建立[J].东北农业大学学报,2008,39(8):104-109.

[19] 李吉霞,葛秀国,王文秀,等.牛乳腺上皮细胞的分离培养[J].黑龙江畜牧兽医,2012(4):4-6.

[20] 宋亚南,陈志伟,刘东武.牛乳腺上皮细胞体外培养体系研究进展[J].生命科学仪器,2008,6:3-8.

[21] PANTSCHENKO A G,YANG T J.Mitogenic responsiveness of caprine mammary epith elial cells to endocrine and cytokine factors[J].Endocrine,1999,10(2):123-130.  

[22] 薛庆善.体外培养的原理与技术[M].北京:科学出版社,2001:387.

[23] AKERS R M.Lactation and the mammary gland ames[M].Iowa:Wiley-Blackwell,2002:45-78.

[24] NEVILLE M C,MCFADDEN T B,FORSYTH I.Hormonal regulation of mammary differentiation and milk secretion[J].Journal of Mammary Gland Biology and Neoplasia,2002,7(1):49-66.  

[25] CIFRIAN E,GUIDRY A J,O'BRIEN C N.Bovine mammary teat and ductal epithelial cell cultures[J].American Journal of Veterinary Research,1994,55:239-246.

[26] SAMANI A A,YAKAR S,LEROITH D,et al.The role of the IGF system in cancer growth and metastasis:overview and recent insights[J].Endocrine Reviews,2007,28(1):20-47.  

[27] BURGOS S A,CANT J P.IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells[J].Domestic Animal Endocrinology,2010,38(4):211-221.  

[28] SHAO Y,WALL E H,MCFADDEN T B,et al.Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland[J].Domestic Animal Endocrinology,2013,44(2):57-69.  
Options
文章导航

/

〈 〉

    联系我们

  • 地址：北京市海淀区圆明园西路2号
    　　　中国农业大学动科动医大楼153室
  • 邮编：100193
  • 电话：010－62817823
  • 网址：http://www.chinajan.com
  • E-mail：cjan@chinajan.com
京公网安备 11010802041926号    京ICP备17050989号-2