欧拉羊胎儿皮肤细胞的培养、保存与鉴定

用胰蛋白酶对欧拉羊胎儿皮肤组织进行热消化分离并培养原代细胞,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代时用液氮保存,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。结果显示,欧拉羊胎儿皮肤细胞呈成纤维型,复苏活力93.5%以上,生长良好,细胞生长曲线呈“S”型,最大增殖量为3.31×10⁵ mL^-1,倍增时间为26.2 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。表明欧拉羊胎儿皮肤细胞分离培养成功,使这一重要种质资源在细胞水平上得以保存。

Culturing, Preservation and Identification of Oula Sheep Embryo Skin Cells

The skin explants of Oula sheep embryo were cultured with trypsinization. The primary cells were purified and preserved with liquid nitrogen after being passaged to the third generation. Observations on vitality, morphology, dynamic growth, microbial contamination and continuous subculturing were carried out. The results showed that the cell line was fibroblast and grow well; the vitality of recovery cell was over 93.5%;the maximum proliferation concentration was 3.31×10⁵ mL^-1 and population doubling time of cells (PDT) was 26.2 h; the tests for bacteria, fungi, virus and mycoplasma were all negative; and the cell line grew normally after being subcultured ten generations. Those showed that the cell line was established successfully, which made the important genetic resource preserved in cell level.