牛胎儿成纤维细胞的培养及性别鉴定

Culture and sex identification of cattle fetal fibroblasts

为了探索和建立牛胎儿成纤维细胞体外分离培养及性别鉴定的技术方法，试验取自怀孕40d的奶牛胎儿耳组织用组织块贴附法进行原代培养和传代培养，用倒置相差显微镜观察成纤维细胞形态；根据牛Y染色体上的性别决定区域（SRY）基因序列设计合成1对PCR引物作为性别鉴定引物，同时根据牛403bp的β-珠蛋白基因序列设计1对引物作为内参照引物，建立PCR反应体系进行性别鉴定。结果表明：分离的成纤维细胞可在体外快速贴壁生长、增殖；阳性对照和第1牛胎儿成纤维细胞系PCR鉴定扩增得到255bp的片段和403bp的β-珠蛋白基因片段，第2，3牛胎儿成纤维细胞系和母牛血液PCR扩增只得到403bp的β-珠蛋白基因片段，通过电泳证明PCR产物255bp的片段为SRY基因片段，说明有255bp扩增带的细胞系为雄性；无255bp扩增带的细胞系为雌性。结果说明该方法所获得的牛胎儿成纤维细胞可在体外稳定培养，利用PCR鉴定牛胎儿性别具有简单、快速、准确的特点，可应用于基因克隆动物研究中体细胞系的早期性别鉴定。

The study explored the method of isolation, cultivation, and sex identification of cattle fetal fibroblasts in vitro. The ear tissue was cultured by tissue explant attachment and observed the cattle fetal fibroblasts by inverted phase contrast microscopy of 40 days cattle fetal. A pair of PCR primers was designed to identify the sex of cattle fetal fibroblast cell lines according to the cattle SRY gene. The isolated fibroblasts could grow and proliferate in vitro. The female sample and cell lines did not amplify the 255 band product ,conversely. The result indicates that PCR of SRY gene is a simple, fast and accurate method to identify the sex of cattle fetal firoblast ceil lines, and can be used in routine study on transgenic cloning animals.