不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响

刘思乐; 康劲翮; 谭支良; 王征

doi:10.3969/j.issn.1006-267x.2016.04.034

动物营养学报 >

2016 , Vol. 28 >Issue 4: 1225 - 1232

DOI: https://doi.org/10.3969/j.issn.1006-267x.2016.04.034

研究简报 Short Communications

不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响

刘思乐 ,
康劲翮 ,
谭支良 ,
王征

展开

1. 中国科学院亚热带农业生态过程重点实验室, 亚热带农业生态研究所, 湖南省农业生态过程重点实验室, 长沙 410125;
2. 湖南农业大学生物科学技术学院, 长沙 410128

收稿日期: 2015-11-03

网络出版日期: 2016-04-11

基金资助

国家自然科学基金项目(31201826);中央驻湘科研机构技术创新发展专项(2013TF3006)

收起

Effects of Different Culture Methods on Growth and Keratin 18 Expression of Ruminal Epithelium Cells of Goats

LIU Sile ,
KANG Jinhe ,
TAN Zhiliang ,
WANG Zheng

Expand

1. Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Key Laboratory of Agro-Ecological Processes in Subtropical Region of Hunan Province, Changsha 410125, China;
2. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China

Received date: 2015-11-03

Online published: 2016-04-11

Fold

摘要

本试验旨在研究不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18(CK18)表达量的影响。采集42日龄山羊的瘤胃上皮组织,分别采用酶消化法和组织块法对其进行体外培养。通过光学显微镜观察原代培养和传代培养阶段的细胞形态,检测第5代山羊瘤胃上皮细胞的生长曲线,并采用细胞免疫荧光法对山羊瘤胃上皮细胞进行鉴定。结果显示:1)经0.25%胰蛋白酶+0.02%乙二胺四乙酸消化获得的原代培养山羊瘤胃上皮细胞于2 d开始贴壁生长,5 d细胞开始明显增多,10 d细胞数量达到最大。2)经组织块法获得的原代培养山羊瘤胃上皮细胞于4 d开始"爬出"组织块,8 d细胞开始明显增多,14 d细胞数量达到最大。3)经免疫荧光染色显示2种方法获得的细胞胞浆内CK18均呈阳性表达且细胞纯度后者明显高于前者。4)组织块法获得的细胞CK18表达量显著高于酶消化法(P<0.05)。综合得出,与酶消化法相比,应用组织块法可成功获得纯度更高的山羊瘤胃上皮细胞。

关键词： 山羊; 瘤胃; 上皮细胞; 原代培养; 分离; CK18

本文引用格式

刘思乐 , 康劲翮 , 谭支良 , 王征 . 不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响[J]. 动物营养学报, 2016 , 28(4) : 1225 -1232 . DOI: 10.3969/j.issn.1006-267x.2016.04.034

Abstract

This research aimed to study the effects of different culture methods on growth and keratin 18(CK18) expression of ruminal epithelium cells of goats. The test proceeded with ruminal epithelium cells of goats aged 42 days, and the cells were cultured using the methods of enzyme digestion and tissue mass culture. The morphology of cells at the stages of primary culture and subculture was observed under the optical microscope, and the growth curve of the fifth generation of ruminal epithelium cells of goat was tested, meanwhile, ruminal epithelium cells were identified using cell immune fluorescence method. The results showed as follows:1) the primary cultured ruminal epithelium cells of goats obtained by digestion of 0.25% trypsin and 0.02% ethylenediamine tetra-acetate began adherent growth on the 2nd day. There was a significant increase in cell number on the 5th day, and the cell number reached a maximum at the 10th day. 2) The primary cultured ruminal epithelium cells of goats obtained by tissue mass culture began‘escape’from the tissue mass on the 4th day. There was a significant increase in cell number on the 8th day, and the cell number reached a maximum at the 14th day. 3) The CK18 in cytoplasm of cells obtained by the 2 methods exhibited a positive reaction by immune fluorescence staining, and cell purity was higher in tissue mass culture method. 4) Tissue mass culture method expressed significantly higher CK18 compared with enzyme method(P<0.05). In conclusion, ruminal epithelium cells of goats were successfully obtained with high purity using the method of tissue mass culture.

Key words： goat; rumen; epithelium cell; primary culture; isolation; CK18

参考文献

[1] STUMPFF F,BONDZIO A,EINSPANIER R,et al.Effects of the Bacillus thuringiensis toxin Cry1Ab on membrane currents of isolated cells of the ruminal epithelium[J].Journal of Membrane Biology,2007,219(1/2/3):37-47.
[2] OBA M,MEWIS J L,ZHINING Z.Effects of ruminal doses of sucrose,lactose,and corn starch on ruminal fermentation and expression of genes in ruminal epithelial cells[J].Journal of Dairy Science,2015,98(1):586-594.

[3] 王佩,范燕茹,金鑫,等.枯草芽孢杆菌对绵羊瘤胃上皮细胞β-防御素-1表达的影响[J].畜牧兽医学报,2015,46(5):760-767.
[4] SCHLAU N,GUAN L L,OBA M.The relationship between rumen acidosis resistance and expression of genes involved in regulation of intracellular pH and butyrate metabolism of ruminal epithelial cells in steers[J].Journal of Dairy Science,2012,95(10):5866-5875.

[5] 卢劲晔,卢炜,刘静,等.IGF-1促进山羊瘤胃上皮细胞增殖的机制[J].南京农业大学学报,2014,37(5):106-110.
[6] 郭立辉.VFA对亚急性瘤胃酸中毒肉牛瘤胃上皮细胞炎性信号通路的影响[D].硕士学位论文.长春:吉林大学,2015.
[7] 成海建,臧坤,刘晓牧,等.不同培养方法对牛前体脂肪细胞增殖分化的影响[J].动物营养学报,2013,25(4):864-869.
[8] XU S W,FU J J,CHEN J W,et al.Development of an optimized protocol for primary culture of smooth muscle cells from rat thoracic aortas[J].Cytotechnology,2009,61(1/2):65-72.
[9] GÁLFI P,NEOGRÁDY S,KUTAS F.Culture of epithelial cells from bovine ruminal mucosa[J].Veterinary Research Communications,1980,4(1):295-300.

[10] 詹康,左晓昕,贡笑笑,等.不同培养模式下奶牛乳腺上皮细胞形态及酪蛋白表达的差异[J].动物营养学报,2015,27(7):2241-2247.
[11] 高玉花,张文秀,吴芳春,等.北京油鸡羊膜上皮细胞的分离、培养及分化潜能的研究[J].畜牧兽医学报,2013,44(2):211-219.
[12] 马瑞丽,张彦明,崔红杰,等.新生仔猪口腔黏膜上皮细胞的体外培养方法[J].畜牧兽医学报,2013,44(9):1445-1453.
[13] 孙志洪,张庆丽,贺志雄,等.山羊瘤胃上皮细胞和空肠黏膜上皮细胞原代培养技术研究[J].动物营养学报,2010,22(3):602-610.
[14] LI H,NIEDERKORN J Y,NEELAM S,et al.Immunosuppressive factors secreted by human amniotic epithelial cells[J].Investigative Ophthalmology & Visual Science,2005,46(3):900-907.

[15] 沈晴昳.不同原代培养方法培养人牙髓细胞的研究[J].天津医药,2011,39(2):139-141.
[16] 詹康,左晓昕,贡笑笑,等.鸡胚小肠上皮细胞的体外分离培养和鉴定[J].动物营养学报,2015,27(7):2150-2156.
[17] 郝白露,杨瑞峰,彭祥炽,等.改良的人脐带间充质干细胞培养方法[J].中国计划生育学杂志,2013,21(1):44-49.
[18] 段永芳,刘继红.家兔阴茎海绵体平滑肌细胞体外培养方法对比研究[J].中国男科学杂志,2002,16(3):197-200.
[19] 何晓琳,李月红,张祥宏,等.人正常食管鳞状上皮不同原代培养方法的比较[J].中国细胞生物学学报,2011,33(6):681-687.
[20] 范燕茹,王佩,金鑫,等.中国驯鹿瘤胃上皮细胞的体外分离培养[J].黑龙江畜牧兽医,2014(11):8-10,15.
[21] 周传丽,刘铮铸,俞英,等.仔猪小肠黏膜上皮细胞体外分离培养及鉴定[J].中国农业科学,2011,44(21):4516-4524.
[22] 李文清,王加启,南雪梅,等.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.
[23] DEHORITY B A,TIRABASSO P A.Effect of feeding frequency on bacterial and fungal concentrations,pH,and other parameters in the rumen[J].Journal of Animal Science,2001,79(11):2908-2912.

Options

文章导航

摘要

本文引用格式

Abstract

参考文献

关于本刊

在线期刊

作者中心

相关单位

联系我们

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献

关于本刊

在线期刊

作者中心

相关单位

联系我们