奶牛子宫内膜上皮细胞体外培养及鉴定

Fostering and Identifying of Dairy Endometrium Epithelial Cell in vitro

试验采用酶消化及机械法相结合的方法培养奶牛子宫内膜上皮原代细胞及传代细胞。酶消化法采用0.1%链蛋白酶于4℃消化16~20h,机械法采用手术刀刮取子宫角内膜。获得原代分离的细胞后,采用胰蛋白酶消化法进行细胞的传代培养。试验应用角蛋白抗体对细胞进行免疫细胞化学鉴定,并对第3代奶牛子宫内膜上皮细胞用MTT法绘制增殖曲线。结果显示,细胞在相差显微镜下呈明显的上皮样细胞形态,细胞传代到第8代时,仍能保持与原代细胞相似的细胞形态特征及生长状态。同时细胞角蛋白染色阳性细胞占80%以上。试验结果表明,应用酶消化和机械法可成功分离并培养数量、活力和纯度均较高的子宫内膜细胞,可操作性强,可在具备基本细胞培养条件的实验室应用推广。

To build up the method of isolating and culturing the dairy endometrial epithelial cells for further study,mechanical method and enzymatic isolation method were used in this study.0.1% pronase was used to soak uterine horns for 16 to 20hat 4℃,and a surgical knife was used to scrape cells from dairy endoemtrium.The dairy endometrial epithelial cells were cultured by trypsin digestion method.The cultured cells were identified by keratin antibody through immunohistochemistry,and the proliferation of the third generation were measured by MTT assay.The results showed that morphology of the cultured cells were similar to epithelia cells by phase-contrast microscope.In addition,the eighth generation still maintained the similar morphology and growth state comparing to the primary generation cells,and the cytokeratin-positive cells ration was more than 80%.The methods used in this study were suitable for culturing the endometrial epithelial cells with highly quantity,activity and purity,it could be popularized in related labs.