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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。
In order to study the small peptide transporter for small peptide transport and absorption mechanism to provide a cell model, in vitro establishment of primary dairy cows intestinal epithelial cells separation method, the use of collagenase type Ⅰ and neutral protease digestion of digestion of dairy cows intestinal digestion by 2% sorbitol was subjected to density gradient centrifugation to remove monocytes, lymphocytes and myocytes; dairy cows’ intestinal epithelial cells were purified by scraping, phase contrast digestion and 96-well plate monoclonal methods, and cell morphology, cytokeratin 18 Immunofluorescent staining and small intestine epithelial cell-specific marker proteins were used to identify cow intestinal epithelial cells. The results showed that: 1) Intestinal digestion of cows’ intestine was induced by collagenase type Ⅰ and neutral protease, and a large number of cell masses could be obtained by 2% sorbitol density gradient centrifugation and adherent to the intestinal mucosa at 48h, but the cell adherent wall was not strong. 2) After 48h, the cell mass further radiated to grow out of the cell, the cell morphology showed typical epithelial cells and paving stone morphology. 5 ~ 7d cells in logarithmic growth phase, a large number of cell proliferation, some of the mixed growth of fibroblasts and intestinal epithelial cells. 3) Cows’ intestinal epithelial cells can be mono-cloned through 96-well plates, and the cells showed uniform paving stone morphology. 4) Cows epithelial keratin 18 was identified as positive and fluoresced green under fluorescent microscope. 5) The expression of E-cadherin, alkaline phosphatase and enteropeptidase, which are specific markers of intestinal epithelial cells in dairy cows, can be detected by RT-PCR, and the method of isolation and culture of dairy cows intestinal epithelial cells has been successfully established.