鸡胚小肠上皮细胞的分离及原代培养研究

Isolation and Primary Culture of Chicken Embryo Small Intestinal Epithelial Cells

取18日龄的SPF鸡胚,分别采用组织块培养法、胰蛋白酶、胶原酶I和嗜热菌蛋白酶消化法分离和体外培养小肠上皮细胞。结果表明:组织块培养法所获得的上皮细胞纯度较低;胰蛋白酶消化后多为单细胞,细胞贴壁和生长能力较弱;胶原酶Ⅰ单独消化50 min或嗜热菌蛋白酶单独消化110 min以及胶原酶Ⅰ、嗜热菌蛋白酶联合消化后均可得到大量隐窝细胞团块,经低速离心去除单细胞、差速贴壁除去成纤维细胞,可获得较纯的上皮细胞。分离的细胞接种培养12～24 h贴壁,48～72 h细胞团中的细胞向四周蔓延,形成一群群的细胞集落,6～7 d细胞汇合成片,成"铺路石样",细胞多呈扁平多角状,椭圆状,单层生长,边界清晰,互不重叠。经形态学观察、扫描电镜鉴定、碱性磷酸酶染色和免疫组化鉴定为上皮细胞。

Eighteenday-old chicken embryos were used to investigate the method for isolation and primary culture of the chicken intestinal epithelial cells.Primary small intestinal epithelial cells from chicken embryo were isolated by means of tissue culture,trypsin digestion,collagenase I digestion and thermolysin digestion,respectively.The results indicated that the epithelial cells obtained by tissue culture method had low purity.They mostly became single cell after trypsin digestion.Cell adhesion and growth were weak.A large number of crypt cell clumps could be obtained by digesting small intestinal tissue with collagenase I for 50 min or thermolysin for 110 min or collagenase Ⅰ,thermolysin protease co-digestion.Single cells and fibroblast cells were removed by low-speed centrifugation and differential velocity adherent,and thus purifed intestinal epithelial cells were obtained.The isolated intestinal epithelial cells were inoculated into cell culture dishes and attached readily to culture dishes within 12-24 h,forming coalescing colonies of epithelium within 48-72 h.Epithelial cells reached confluence within 6-7 days resembling paving stones.The cultured cells were identified as small intestinal epithelial cells by morphological observation,scanning electron microcope observation,alkaline phosphatase staining and immunohistochemical characterization.