奶牛乳腺上皮细胞模型的建立与鉴定

Establishment and identification of bovine mammary epithelial cells model

为了建立奶牛乳腺上皮细胞模型,并对建立的细胞模型生物学特性进行鉴定,试验采用组织块培养法和乳汁分离法分离培养奶牛乳腺上皮细胞,利用差时胰酶消化法纯化奶牛乳腺上皮细胞,通过测定生长曲线、细胞群体倍增时间、核型、奶牛乳腺上皮细胞骨架蛋白-角蛋白等对所培养细胞进行鉴定,以确定其为奶牛乳腺上皮细胞,并用金黄色葡萄球菌侵染奶牛乳腺上皮细胞,用流式细胞仪(Annexin V/PI双染法)检测奶牛乳腺上皮细胞的凋亡率和坏死率。结果表明:显微镜下观察可见纯化的乳腺上皮细胞为典型上皮细胞形态,呈多角形或乳滴形。细胞生长曲线呈"S"形;细胞群体倍增时间最短为36.6 h;细胞核型正常(2 n=60,XX);细胞特异性蛋白-角蛋白表达呈阳性,金黄色葡萄球菌感染奶牛乳腺上皮细胞3 h,Annexin V/PI双染法检测发现感染组细胞凋亡率和坏死率均明显升高,差异极显著(P<0.01)。说明试验成功建立了奶牛乳腺上皮细胞模型。

The present study was carried out to establish bovine mammaryepithelial cells （BMECs）, and identify their biological characteristics. Two methods of tissue mass cell culture and milk separation were chose for preparation of BMECs. The purification of the isolated BMECs was performed through the trypsin digestion at different time points. The biology characteristics of the isolated BMECs were determined, involving in the growth curves, cell population doubling time, keleton protein - keratin, and karyotype. After the isolated BMECs were challenged by Staphylococcus aureus, the apoptosis level of the isolated BMECs was examined by using Flow Cytometry （Annexin V/PI） . The results demonstrated that the isolated BMECs exhibited a typical epithelial ceils, showing the polygonal or emulsion droplet; the cell growth curve exhibited the ＂S＂ shape ; the shortest doubling time of cell population was at 36.6 h ; karyotype of the isolated BMECs was revealed to be normal （ 2n = 60, XX） ; cytokeratin expression of the isolated BblECs was positive; the apoptosis level of the isolated BMECs was very significantly up - regulated （P 〈 0.01 ）. These data indicated that the BMECs model were successfully established in our study.