Experimental Methods

Culture and Identification of Jejunal Epithelial Cells Isolated from Neonatal Rabbits in Vitro

  • FAN Bin ,
  • YU Bing ,
  • TIAN Gang ,
  • ZHANG Kai ,
  • LIU Hanzhong
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  • 1. Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130, China;
    2. Key Laboratory of Animal Disease-Resistant Nutrition of Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China;
    3. Sichuan Academy of Grassland Sciences, Chengdu 611743, China

Received date: 2013-12-13

  Online published: 2014-05-30

Abstract

This study was aimed to establish a method to culture jejunal epithelial cells (JEC) isolated from rabbits, and provide an in vitro model for the further study of the effects of exogenous substances on rabbit intestinal epithelial structure and mechanism. Newborn New Zealand white rabbits (1 to 3 days of age) were used, jejunum was removed and JEC were isolated by the application of digestive juice mixed with pronase E, collagenase Ⅳ and dithiothreitol (DTT), and the crypt cell aggregates were enriched by density gradient centrifugation of 2% sorbitol. The rabbit JEC were purified by differential digestion method and differential velocity adherent method. Cells were identified by morphological observation, alkaline phosphatase staining, cytokerain-8 immunofluorescence staining and the gene expression analyses of enterocyte markers. The results showed that the application of digestive juice mixed with pronase E, collagenase Ⅳ and DTT could successfully get a lot of rabbit JEC aggregates, which readily adhered to culture dishes within 12 hours and grown to around radially, growing cells showed typical triangular or polygonal, reached confluence within 5 to 7 days, and showed monolayer and non-overlapping; cells could be passaged and purified; cells were stained positive for alkaline phosphatas staining and cytokerain-8 immunofluorescence staining; enterocyte markers, such as fatty acid binding protein 2, enterokinase, E-cadherin, claudin-4, were expressed in JEC. These results show that an ideal method for isolating and culturing epithelial cells of rabbit jejunum is established.

Cite this article

FAN Bin , YU Bing , TIAN Gang , ZHANG Kai , LIU Hanzhong . Culture and Identification of Jejunal Epithelial Cells Isolated from Neonatal Rabbits in Vitro[J]. Chinese Journal of Animal Nutrition, 2014 , 26(6) : 1570 -1578 . DOI: 10.3969/j.issn.1006-267x.2014.06.018

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