鸡胚盲肠上皮细胞原代培养与鉴定

Study on Primary Culture and Identification of Cecum Epithelial Cells from Chicken Embryo

为研究鸡E.tenella的宿主细胞——盲肠上皮细胞体外培养技术,为E.tenella损伤机制及抗球虫药的研究提供体外模型,分别用胰蛋白酶法、胶原酶Ⅰ法、嗜热菌蛋白酶法、嗜热菌蛋白酶+胶原酶Ⅰ法和中性蛋白酶Ⅰ+胶原酶Ⅺ法分离纯化原代鸡胚盲肠上皮细胞,通过测定细胞活力、细胞团块比例、总细胞产量和细胞团块产量,筛选出鸡胚盲肠上皮细胞最佳分离方法,并进行了纯化和培养,对培养细胞进行了鉴定。结果表明:嗜热菌蛋白酶消化、低速离心去除单细胞、差速贴壁除去成纤维细胞,为最佳分离和纯化盲肠上皮细胞的方法;分离纯化的细胞接种后分别于第3-5天、第10-11天进入对数生长期,可存活14 d以上;经形态学观察、碱性磷酸酶染色、扫描电镜鉴定为盲肠上皮细胞,所分离的细胞培养至第4、7、11天时上皮细胞比例分别为81.67%、84.33%和72.00%。用该法可获得纯度较高的原代鸡胚盲肠上皮细胞。

To establish an in vitro model for studying the injury mechanism of E.tenella,in vitro culture techniques of cecum epithelial cells,the host cells of E.tenella in chicken,were studied.Primary cecum epithelial cells from chicken embryo were isolated by the digestion of trypsin,collagenaseⅠ,thermolysin,thermolysin/collagenaseⅠ and dispaseⅠ/collagenaseⅪ,respectively.The best method for separating chicken embryo cecum epithelial cells was screened out by determining the cell viability,proportion of cell aggregates,cell total yield and cell aggregates yield.Thereafter cell purification,culture and identification were performed.The results showed that the best methods for isolating and purifying cecum epithelial cells were digesting the cecum from chicken embryo with thermolysin,then removing single cells by low-speed centrifuge and fibroblast cells by differential velocity adherent.The isolated and purified cells obtained using the above method stayed in logarithmic growth phase from the 3rd to 5th day and from the 10th to 11th day after planting,respectively,and could survive for more than 14 days.The cultured cells were identified as cecum epithelial cells by morphological observation,alkaline phosphatase staining and scanning electron microscope observation.The proportions for epithelial cells at the 4th,7th and 11th day were 81.67%,84.33% and 72.00%,respectively,which implied that the primary cecum epithelial cells with high purity could be obtained from chicken embryo by this method.