Abstract: 【Objective】 To elucidate the pathogenic mechanism of enterotoxigenic Escherichia coli F18 (ETEC F18), 3 cell lines with GG, GA, AA genotypes of the candidate gene FUT1 at CDS 307 (G→A) were established using piglets’ small intestinal mucous membrane.【Method】In this study, fetal rats and neonatal piglets were firstly used as preliminary experiment materials to compare the effectiveness of two digestion methods for isolating intestinal mucous membrane cells. The first digestion method was that the fetal rat intestine was cutted mechanically and then digested with prepared mixture of collagenase XI and dispase I. The senod one was that the small intestine of neonatal pig was directly filled with the mixture. Removal of the fibroblast-cell- contamination is one of the troublesome things during the process of intestinal cells primary culture in vitro. In this study, purification of the epithelium was facilitated by using a simple partial digestion method upon the contamination of the fibroblast cells. Finally, the purified primary small intestine epithelial cells were identified using electron microscopy and CK18 immunofluorescence staining.【Result】The results showed that the second digestion way was better than the first one. The neonatal Large White that was born within 12 h was picked and its small intestine was cutted into segments after laparotomizing. Isolation of the epithelia and preservation of its three-dimensional integrity was achieved via collagenase/dispase digestion technique. The photographs of electron microscopy and CK18 immunofluorescence staining showed that the purity of the epithelial cells was above 90%. The entire period of growing pig small intestinal epithelium in primary cultures showed that before the seventh generations of sub-culturing the cells grew well, which showed the typical feature of epithelial cells and the monolayer was pavement-like. After the nineth generations, these untreated cells reached arrest of growth and vacuolization.【Conclusion】Small intestinal epithelial cells with GG and GA genotypes of FUT1 gene were primarily cultured in vitro. In the present study, an ideal method for isolating and culturing epithelial cells of piglets’ small intestinal mucous membrane was established, which laid a good foundation for establishing cell lines of piglet epithelial cell.

Key words: piglet, mucous membrane of small intestine, epithelial cell, in vitro isolation and culture, cell identification