奶牛小肠上皮细胞的原代培养和鉴定

Study on primary culture and identification of bovine intestinal epithelial cells

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。

This experiment was designed to study the small peptides transporter and construct cell model based on the principle of small peptides transport and absorption.Bovine small intestine tissue were digested with mixture of collagenase Ⅰ and dispase.The removal of mononuclear cells,lymphocytes and muscle cells were performed by density gradient centrifugation with 2% D-Sorbitol.Purification of bovine intestinal epithelial cells was done by curettage,phase contrast digestion and 96 orifice plate monoclonal method.The methods of morphological observation,cytokeratine 18 immunofluorescence and gene expression analysis of intestinal epithelial cell markers were used to identify the bovine intestinal epithelial cells.The results showed that：1） Using mixture of collagenase Ⅰ and dispase to digest bovine small intestine tissue and through 2% D-Sorbitol density gradient centrifugation obtained a large number of cell mass and the cells adheren in 48 h,but cell adherence was not strong;2） After 48 h,the cell mass grew out of the cells the cell morphology was typical epithelial cells and paving stones,and the cells were in logarithmic growth phase,abundant in growth and a part of fibroblasts and small intestinal epithelial cell mixed growth in 5-7 d;3） Through the 96 hole plate method,the bovine intestinal epithelial cells and the cells showed homogeneous paving stones were obtained;4） The cytokeratine 18 immunofluorescence was positive and emitted green fluorescence under the fluorescence microscope;5） Though RT-PCR,the expressions of the small intestinal epithelial cell specific marker protein E-cadherin,alkaline phosphatase and intestinal peptide were detected.The above results show that this study successfully established the method of isolation and culture of bovine intestinal epithelial cells and for further study the absorption mechanism of small peptide transport.It also provided an ideal cell model for differentiation of mucosal immunity M cell.