奶牛小肠上皮细胞的原代培养和鉴定

发布时间：2018-04-15 07:43

本文选题：奶牛 + 小肠上皮细胞　；参考：《中国农业大学学报》2017年06期

【摘要】：为研究小肽转运蛋白对小肽转运和吸收机制提供细胞模型,在体外建立原代奶牛小肠上皮细胞分离的方法,采用Ⅰ型胶原酶和中性蛋白酶混合消化液对奶牛小肠组织进行消化,通过2%山梨醇进行密度梯度离心以去除单核细胞、淋巴细胞和肌细胞;用刮除法、相差消化法和96孔板单克隆方法来纯化奶牛小肠上皮细胞,从细胞形态学、细胞角蛋白18免疫荧光染色和小肠上皮细胞特异性标志蛋白来鉴定奶牛肠上皮细胞。结果表明:1)采用Ⅰ型胶原酶和中性蛋白酶联合消化奶牛小肠组织,通过2%山梨醇密度梯度离心可以获得大量的细胞团且48h发生贴壁,但是细胞贴壁不牢。2)48h之后细胞团进一步向外辐射生长出细胞,细胞形态呈现典型的上皮细胞和铺路石形态。5~7d细胞处于对数生长期,细胞大量增值,一部分成纤维细胞与小肠上皮细胞夹杂生长。3)通过96孔板能够单克隆奶牛小肠上皮细胞,细胞呈现均一的铺路石形态。4)奶牛小肠上皮细胞角蛋白18鉴定为阳性,荧光显微镜下能够发出绿色荧光。5)通过RT-PCR能够检测到奶牛小肠上皮细胞特异性标志蛋白E-钙黏蛋白、碱性磷酸酶和肠肽酶的表达,成功建立了奶牛小肠上皮细胞的分离和培养方法。
[Abstract]:In order to study the mechanism of small peptide transporter on the transport and absorption of small peptides, a method of isolation of primary small intestinal epithelial cells from dairy cattle was established in vitro. The mixed digestive fluid of type I collagenase and neutral protease was used to digest the small intestinal tissue of dairy cattle.Density gradient centrifugation with 2% sorbitol was used to remove monocytes, lymphocytes and myocytes, and bovine small intestinal epithelial cells were purified by scraping method, phase difference digestion method and 96-well plate monoclonal method.Cytokeratin 18 immunofluorescence staining and small intestinal epithelial specific marker protein were used to identify bovine intestinal epithelial cells.The results showed that a large number of cell clusters could be obtained by 2% sorbitol density gradient centrifugation by digesting the small intestine tissue with type I collagenase and neutral protease.However, after 48 hours of cell attachment, the cell mass further radiated out, and the cell morphology showed typical epithelial cells and paving stone morphology. The cells were in logarithmic growth stage, and the cells increased in a large number.Some fibroblasts and small intestinal epithelial cells mixed with growth. 3) through 96 well plate can monoclonal bovine small intestinal epithelial cells, the cells show a uniform paving stone morphology. 4) Cow small intestinal epithelial cell keratin 18 was identified as positive.The expression of E-cadherin, alkaline phosphatase and enteropeptidase was detected by RT-PCR.The method of isolation and culture of bovine intestinal epithelial cells was established successfully.
【作者单位】：扬州大学动物科学与技术学院;
【基金】：国家自然科学基金资助项目(31572430)
【分类号】：S823

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