摘要
【目的】比较4种小鼠肠黏膜上皮细胞(Intestinal epithelial cells,IECs)分离方法的分离效果,建立小鼠IECs的有效分离培养方法,获得小鼠IECs原代细胞,为后续研究做准备。【方法】分别采用组织块培养法、嗜热菌蛋白酶消化法、胶原酶Ⅺ和中性蛋白酶Ⅰ联合消化法以及胶原酶Ⅰ和中性蛋白酶Ⅵ联合消化法共4种方法分离小鼠IECs并培养,通过细胞免疫组织化学法和细胞免疫荧光法对分离的IECs进行细胞特异性鉴定,比较4种方法的分离效果。【结果】组织块培养法所获IECs活力好,增殖能力强,但成纤维细胞污染较严重,细胞纯度低;胶原酶Ⅰ和中性蛋白酶Ⅵ分离法获得的IECs数量少且细胞增殖能力弱;嗜热菌蛋白酶消化法及胶原酶Ⅺ与中性蛋白酶Ⅰ联合消化法所获得的小鼠IECs纯度较高,原代培养时增殖能力稍弱于组织块培养法,但传代后增殖能力趋于稳定。通过细胞免疫组织化学法和细胞免疫荧光法对分离的细胞进行鉴定,结果表明,分离的细胞多数为小鼠IECs。【结论】嗜热菌蛋白酶消化法及胶原酶Ⅺ与中性蛋白酶Ⅰ联合消化法均适合肠道上皮细胞的分离和培养。
【Objective】 Separation results of four primary culture methods for mouse intestinal epithelial cells were compared to choose and establish utility separation culture method for obtaining mouse IECs and preparing for future research.【Method】 The four methods,tissue fractional cultivation,thermolysin enzyme digestion,association digestion of collagenase XI and dispase I,and association digestion collagenase I and dispaseⅥ were used to separte and culture mouse IECs.The separation results were compared by the cell immunohistochemistry method and cell immunofluorescence method to identify the cell specificity of the separated IECs.【Result】 The results showed that the intestinal epithelial cells obtained by tissue culture method had the highest proliferation ability and better viability,but with serious fibroblasts pollution and lower purity.The collagenase I and neutral proteaseⅥ method only obtained a few intestinal epithelial cells with lower proliferation.The rmolysin digestion method and the joint digestion method of collagenase XI and neutral protease I obtained intestinal epithelial cells with higher purity and the proliferation ability was slightly weaker than the tissue culture method.However,the proliferation ability was more stabilized.The results of cell specificity of the separated IECs by the two methods using the cell immunohistochemistry method and cell immunofluorescence method showed that most of the separated cells were IECs.【Conclusion】 Thermolysin digestion method and Collagenase Ⅺ and Dispase Ⅰ combined digestion method are suitable for isolating and cultivating Intestinal epithelial cells.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2013年第5期25-31,共7页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31001019)
安徽省自然科学基金项目(11040606M91)
关键词
小鼠
小肠上皮细胞
原代培养
组织块分离培养法
酶学分离培养法
mouse
mouse intestinal epithelial cells
primary culture
tissue fractional cultivation
enzymology fractional cultivation
