仔猪小肠黏膜上皮细胞体外分离培养及鉴定

 【目的】阐明产肠毒素大肠杆菌F18（ETEC F18）引发仔猪腹泻的机理，针对其候选基因FUT1 的CDS区第307位点处的突变（G→A），建立3个仔猪小肠黏膜上皮细胞系（GG、GA和AA）。【方法】首先以胎鼠和仔猪为试验材料，确定小肠上皮细胞的最佳分离方法。采用XI型胶原酶和I型中性蛋白酶的联酶混合液，将胎鼠小肠组织机械剪碎后用联酶进行消化和直接将联酶灌注到仔猪小肠腔内消化的两种方法进行比较。针对仔猪小肠黏膜上皮细胞体外培养过程中成纤维细胞污染难以消除的问题，本研究采用局部画圈法逐步去除成纤维细胞。最后，用电镜和CK18免疫荧光染色对纯化后的原代仔猪小肠黏膜上皮细胞进行鉴定。【结果】两种小肠上皮细胞分离效率对比表明，后一种消化方式更为有效。进而取出生12 h内未吃初乳的大白仔猪，剖腹剪取其小肠段，使用联酶混合液灌注消化分离得到形态完整、容易贴壁生长的小肠绒毛细胞团。电镜和CK18免疫荧光染色结果均显示，小肠黏膜上皮细胞纯度达90%以上。经纯化处理，最后获得了FUT1基因型为GG和GA的两种小肠黏膜上皮细胞。仔猪原代小肠黏膜上皮细胞培养周期显示，7代前细胞生长状况良好，形态为典型的上皮细胞样，单层细胞呈铺路石排列，培养至第9代，细胞出现生长停滞、胞体空泡化等现象。【结论】最终获得了FUT1基因型为GG和GA的仔猪原代小肠黏膜上皮细胞。建立了仔猪小肠黏膜上皮细胞分离及培养方法，为后续的细胞建系工作奠定了良好的基础。

Isolation and Identification of Piglets' Small Intestinal Mucous Membrane Epithelial Cells

【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.