欧拉羊胚肾细胞的分离培养和鉴定

Separation,Culture and Identification of Embryo Kidney Cells in Oula Sheep

[目的]分离培养并鉴定欧拉羊胚肾细胞,为该品种基因组文库构建和遗传多样性研究提供生物学材料。[方法]用胰蛋白酶热消化法分离并培养欧拉羊胚肾原代细胞,通过差速消化和差速贴壁法纯化成纤维细胞,扩大培养至第3代用液氮保存,细胞复苏后进行活力、形态、生长曲线、微生物污染检测和连续传代培养试验。[结果]欧拉羊胚肾细胞呈成纤维型,复苏活力90%以上,生长良好,生长曲线呈"S",最大增殖浓度为4.61×105/ml,倍增时间26 h;细菌、真菌、病毒、支原体检测呈阴性,连续传代培养在10代内生长正常。[结论]欧拉羊胚肾细胞分离培养成功,使这一重要种质资源在细胞水平上得以保存。

[ Objective ] The study aimed to separate,culture and identify the embryo kidney ceils in Oula sheep and provide the biological mate- rial for the establishment of the genome library and genetic diversity of this variety. [ Method ] The primary cells of the embryo kidney in Oula sheep were separated and cultured with the trypsin digestion method,purified into the fibroblasts through the differential digestion and differential adherence isolation methods and preserved with the liquid nitrogen when expanding the culture to the third generation. The observations on the vi- tality, morphology and dynamic growth, the detection on the microbial contamination and the test on the continuous subculture were carried out af- ter the recovery of the cells. [ Result ] The cells of embryo kidney in Oula sheep were fibroblast and the vitality of the recovery cell was over 90% ; the ceils grown well ,with the growth curve showing ＂S＂ type ,the maximum proliferation concn, of 4.61 ~ 105/ml and the population dou- bling time （PDT） of 26 h; the detection on the bacteria, fungi,virus and mycoplasma showed all negative and the ceils grown normally after being subcultured within 10 generations. [ Conclusion] The cells of the embryo kidney in Oula sheep were separated and cultured successfully, which