新生仔猪口腔黏膜上皮细胞的体外培养方法

doi:10.11843/j.issn.0366-6964.2013.09.016

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (9): 1445-1453.doi: 10.11843/j.issn.0366-6964.2013.09.016

新生仔猪口腔黏膜上皮细胞的体外培养方法

马瑞丽^1,2，张彦明^1*，崔红杰¹，郭抗抗¹

(1. 西北农林科技大学动物医学院，杨凌 712100; 2. 西北农林科技大学生命科学学院，杨凌 712100)

收稿日期:2013-03-18 出版日期:2013-09-23 发布日期:2013-09-23
通讯作者: 张彦明，教授，主要从事分子病原学与免疫学方面的研究，E-mail:ylzhangym@sohu.com
作者简介:马瑞丽(1976-)，女，陕西临潼人，讲师，博士生,主要从事分子病原学与免疫学研究，E-mail: maruili2006@126.com
基金资助:
陕西省13115科技创新工程重大科技专项(2010ZDKG-71)

The Culturing Method of Neonatal Piglet Oral Mucosal Epithelial Cells in vitro

MA Rui-li^1,2, ZHANG Yan-ming^1*, CUI Hong-jie¹, GUO Kang-kang¹

(1. College of Veterinary Medcine, Northwest A&F University, Yangling 712100, China;2. College of life Sciences, Northwest A&F University, Yangling 712100, China)

Received:2013-03-18 Online:2013-09-23 Published:2013-09-23

摘要/Abstract

摘要：

为了建立仔猪口腔黏膜上皮细胞（oral mucosal epithelial cells, OMEC）的体外培养方法，取新生仔猪的口腔颊膜组织，分别采用组织块培养法、胰蛋白酶消化法、DispaseⅡ和胰蛋白酶联合消化培养法、DispaseⅡ处理联合组织块培养法，对口腔黏膜上皮细胞进行原代和传代培养，用倒置显微镜观察培养细胞形态学特征及生长情况，对传代培养的OMEC，采用间接免疫荧光法检测广谱细胞角蛋白的表达情况，并进行透射电镜观察，用MTT法测定细胞的生长曲线，流式细胞仪检测其细胞周期。结果表明，在采用相同培养基条件下, 采用组织块培养法、胰蛋白酶消化法不能成功培养仔猪OMEC，用DispaseⅡ和胰蛋白酶联合消化法培养的细胞不易贴壁，培养困难，采用DispaseⅡ处理联合组织块培养法培养的细胞增殖能力强，细胞纯度高，可连续传到10～11代，经鉴定具有OMEC特征。本试验成功建立了仔猪口腔黏膜上皮细胞的原代及传代培养方法，为进一步研究OMEC的永生化及猪口蹄疫致病机理和疫苗提供理想实验材料。

Abstract:

The aim of this study was to establish the culture method of piglet oral mucosal epithelial cells in vitro. Piglet buccal cells were cultured and subcultured with the direct explant technique, trypsin digestion, combined method of DispaseⅡand trypsin digestion, and DispaseⅡ combined with explant technique. Morphological characteristics were observed under the light microscope and transmission electron microscope (TEM). For second cultures, the expression of cytokeratin was detected by indirect immunofluorescence, cell growth curve was measured by MTT method，cell cycle distribution was evaluated by flow cytometry. The results showed that in the same culture medium the explant technique and enzymatic method were not successful, the cells obtained by the method of combination of dispaseⅡand trypsin digestion were difficult to attach and grow, but the cells obtained by the combination of DispaseⅡ and explant technique proliferated quickly, and were of high purity, which can be passaged continuously till to the 10-11 generations, and the cells were identified with the characteristics of oral mucosal epithelial cells. The culture method of piglet oral mucosal epithelial cells in vitro has been established successfully, which may provide ideal research material for study the immortalization of oral mucosal epithelial cells, swine foot-and-mouth disease (FMD) pathogenesis and its vaccine.

中图分类号:

Q813.1

马瑞丽，张彦明，崔红杰，郭抗抗. 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.

MA Rui-li, ZHANG Yan-ming, CUI Hong-jie, GUO Kang-kang. The Culturing Method of Neonatal Piglet Oral Mucosal Epithelial Cells in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(9): 1445-1453.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2013.09.016

https://www.xmsyxb.com/CN/Y2013/V44/I9/1445

参考文献

［1］INOUE H，OSHIMA H，MATSUZAKI K，et al. Application for regenerative medicine of epithelial cell culture-vistas of cultured epithelium［J］. Congenit Anom (Kyoto)，2006, 46(3):129-134.

［2］ZUBAIR M, EKHOLM A, NYBOM H，et al. Effects of Plantago major L. leaf extracts on oral epithelial cells in a scratch assay［J］. J Ethnopharmacol, 2012, 141(3):825-830.

［3］VILLAR C C, CHUKWUEDUM ANIEMEKE J, ZHAO X R, et al. Induction of apoptosis in oral epithelial cells by Candida albicans［J］. Mol Oral Microbiol, 2012, 27(6)：436-448.

［4］MADHIRA S L, VEMUGANTI G, BHADURI A, et al.Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction［J］. Mol Vis, 2008，14:189-196.

［5］LUO X, OKUBO T, RANDELL S, et al. Culture of endodermal stem/progenitor cells of the mouse tongue［J］. In Vitro Cell Dev Biol Anim, 2009, 45(1-2): 44-54.

［6］ARENHOLT-BINDSLEV D, JEPSEN A, MACCALLUM D K, et al. The growth and structure of human oral kerationocytes in culture［J］. J Invest Dermatol, 1987, 88(3): 314-319.

［7］NAKAMURA K，ENDO K，COOPER L J，et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane［J］. Invest Ophthaomol Vis Sci，2003，44(1)：106-116.

［8］ODA D, BIGLER L, LEE P, et al. HPV immortalization of humnan oral epithelial cells: a model for carcinogenesis［J］. Exp Cell Res, 1996, 226(l):164-169.

［9］ODA D，SAVARD C E，ENG L，et al. Reconstituted human oral and esophageal mucosa in culture［J］. In Vitro Cell Dev Biol Anim, 1998，34(1)：46-52.

［10］GROGER S, MICHEL J, MEYLE J. Establishment and characterization of immortalized human gingival keratinocyte cell lines［J］. J Periodontol Res, 2008, 43(6): 604-614.

［11］FUJII S, MAEDA H, WADA N, et al. Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer［J］. Cell Tissue Res, 2006, 324(1):117-125.

［12］THONEMANN B, SEHMALZ G. Immortalization of bovine dental papilla cells with simian virus 40 large t antigen［J］. Areh Oral Biol, 2000, 45(10):857-869.

［13］KIBE T, KISHIDA M, KAMINO M, et al. Immortalization and characterization of normal oral epithelial cells without using HPV and SV40 genes［J］. Oral Sci Int, 2011, 8(1):20-28.

［14］于世凤，高岩.口腔组织学与病理学［M］.北京：北京大学医学出版社，2005:119-126.

［15］STENN K S, LINK R, MOELLMANN G, et al. Dispase, a neutral protease from Bacillus polymyxa,is a powerful fibronectinase and type IV collagenase［J］. J Invest Dermatol, 1989, 93(2):287-290.

［16］SCHAEFER B M, WALLICH R, SCHMOLKE K, et al. Immunohistochemical and molecular characterization of cultured keratinocytes after dispase-mediated detachment from the growth substratum［J］. Exp Dermatol, 2000, 9: 58-64.

［17］BAYAR G R, AYDINTUG Y S, GULSES A, et al. A pilot study of the primary culture of the oral mucosa keratinocytes by the direct explant technique［J］. OHDM, 2011, 10(2): 88-92.

［18］KEDJARUNE U, PONGPRERACHOK S, ARPORNMAEKLONG P, et al. Culturing primary human gingival epithelial cells: comparison of two isolation techniques［J］. J Craniomaxillofac Surg, 2001，29(4)：224-223.

［19］SDEK P，ZHANG Z Y，CAO J，et al. Alteration of cell-cycle regulatory proteins in human oral epithelial cells immortalized by HPV16 E6 and E7［J］. Int J Oral Maxillofac Surg, 2006, 35(7): 653-657.

［20］洪海霞.永生化猪脐静脉血管内皮细胞系的建立及其生物学特征分析［D］.杨凌：西北农林科技大学，2007.

［21］IGARASHI M, IRWIN C R, LOCKEI M, et al. Construction of large area organotypical cultures of oral mucosa and skin ［J］. J Oral Pathol Med, 2003，32：422-430.

［22］FORMANEK M, MILLESI W, WILLHEIM M, et al. Optimized growth medium for primary culture of human oral keratinocytes［J］. Int J Oral Maxillofac Surg，1996，25(2)：157-160.

[1]	陈奡蕾, 黄德如, 安娅菲, 李守军. 动物肠类器官培养技术[J]. 畜牧兽医学报, 2023, 54(7): 2743-2750.
[2]	田青,王洪荣,王梦芝. 氢化可的松对奶牛乳腺上皮细胞酪蛋白合成的影响[J]. 畜牧兽医学报, 2014, 45(10): 1663-1670.
[3]	吕婧玉，许晓婷，陈晓佩，翟明胜，冯建昆，于文浩，王新庄. 5-aza-dC体外诱导兔骨髓间充质干细胞分化为胰岛细胞的研究[J]. 畜牧兽医学报, 2014, 45(9): 1538-1543.
[4]	廖庆红，丁培阳，赵丹丹，王爱华，齐雪峰. 山羊子宫内膜细胞与性腺激素对uNK细胞分泌活性的调节作用[J]. 畜牧兽医学报, 2013, 44(6): 866-870.

新生仔猪口腔黏膜上皮细胞的体外培养方法

The Culturing Method of Neonatal Piglet Oral Mucosal Epithelial Cells in vitro

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 4

编辑推荐

Metrics

本文评价