奶牛乳腺上皮细胞和成纤维细胞的体外培养与鉴定

为体外培养纯化出稳定的奶牛乳腺上皮细胞和成纤维细胞,试验通过外科手术的方法取妊娠后期或泌乳期的荷斯坦奶牛乳腺组织,分离乳腺腺泡,用组织块法体外培养奶牛乳腺细胞,应用差时胰酶消化法和差速贴壁法将奶牛乳腺上皮细胞和成纤维细胞分别纯化出来,并用免疫组化的方法对细胞的纯度进行鉴定。结果表明:纯化的奶牛乳腺上皮细胞多为多角形,细胞核呈圆形或椭圆形,核仁清晰可见,多呈鹅卵石样或铺路石样生长,并可分泌乳滴,角蛋白-18反应阳性,波形蛋白反应阴性;纯化的奶牛乳腺成纤维细胞多为长梭形,呈旋涡状或放射状生长,角蛋白-18反应阴性,波形蛋白反应阳性;经纯化后2种细胞的纯度均可达95%以上,可满足后续试验的要求。

Identification and in vitro culture of bovine mammary epithelial cells and fibroblast

To obtain stabile purified bovine mammary epithelial cells and fibroblast in vitro,the mammary tissue samples were collected from Holstein cows during late gestation or lactation by surgery.After isolated the mammary alveoli,the mammary cells were cultured by tissue explants method in vitro.Then bovine mammary epithelial cells and fibroblasts were purified and cultured separately at different time by trypsin-EDTA digestion and adherent method,and the purity of the cells were identified by immunocytochemical method.The results showed that purified bovine mammary epithelial cells were mainly in polygonal,with round or oval nucleus,the nucleolus were visible and clear,which mainly in cobblestone or paving stone-like growth,and also could secret milk drop,with a positive reaction to cytokeratin-18 and a negative reaction to vimentin,while purified bovine mammary fibroblasts were mainly in spindle,in spiral or radial-shaped growth,with a negative reaction to cytokeratin-18 and a positive reaction to vimentin.After purification,the purity of both cells could exceed ninety-five percent,which could meet the requirement of the follow-up experiments.