水牛乳汁中乳腺上皮细胞的分离培养及鉴定

Isolation,Culture and Identification of Buffalo Mammary Epithelial Cells from Milk

试验旨在从水牛(Bubalus bubalis)乳汁中分离培养水牛乳腺上皮细胞并对其进行鉴定,建立经济、便捷的水牛乳腺上皮细胞分离方法。通过无菌收集高产奶水牛泌乳后期乳汁,将其与PBS按2∶1(V/V)混匀离心;去除上层乳脂,取带少量上清的沉淀,与PBS按上述比例混匀再次离心;将脂肪层去除干净,取沉淀上方1 mL上清及沉淀分别接种于细胞培养皿。通过细胞形态、免疫荧光、生长曲线测定、细胞接种活力、RT-PCR等对分离的细胞进行鉴定。结果表明,试验成功从水牛乳汁的上清和沉淀中分离获得了水牛乳腺上皮细胞,上清部分细胞和杂质较少;而沉淀部分则细胞和杂质较多。细胞呈典型鹅卵石样,无成纤维细胞,细胞活力好,多处于分裂期。当细胞增殖高度融合时,出现乳头状结构和分层生长现象。经免疫荧光鉴定,分离获得的水牛乳腺上皮细胞表达上皮细胞标志蛋白——细胞角蛋白18。水牛乳腺上皮细胞的生长曲线呈S型,符合一般生物学规律。细胞接种存活率的测定结果显示,细胞活力好、贴壁能力强。经RT-PCR检测,水牛乳腺上皮细胞表达乳蛋白及乳脂合成相关基因。从乳汁中分离水牛乳腺上皮细胞并对其进行鉴定,成功建立了水牛乳汁分离乳腺上皮细胞的方法,为研究水牛泌乳机制、改善乳品质、提高产奶量奠定基础。

This study was aimed to set up a simple,economic and pure method to culture and identify buffalo mammary epithelial cell in vitro,which were collected from the fresh milk.The milk was collected in the late lactation period of the high yield milk buffalo,then it was mixed with the PBS in the ratio of2∶1and centrifugated.A few suspernatants and the pellets were resuspended with the PBS when the milk fat were decanted,then recntrifuged.The final pellets and1mL of suspernatants were seeded in petri dish without fat,respectively.The isolated cells were mammary epithelial cells which were identified by cell morphology,immunofluorescence,growth curve,cell viability and RT PCR.The buffalo mammary epithelial cells were successfully isolated from both the pellets and supernatants.The cells and impurities were less in the supernatants than that in the pellets.The cells were cobblestone like without fibroblasts and most in cell division.In the post confluent culture,mammary epithelial cells formed dome structures and layer separated growth.The cells expressed cytokeratin18was identified by immunofluorescence which was the marker of mammary cells.The cell growth curve and cell survival rate were measured.The results showed that the buffalo mammary epithelial cells were activity and easy to attach.The mammary epithelial cells were expressed of milk protein and milk fat related genes detected by RT PCR.The buffalo mammary epithelial cell line were successfully isolated and identified from fresh milk,which estabilished foundation for the study of the mechanism of lactation,the amelioration of the quality of milk and the improvement of milk yield of buffalo.