胎牛子宫类器官及内膜上皮细胞的分离培养

Isolation and culture of organs and endometrial epithelial cells of fetal bovine uterine

建立胎牛子宫类器官及内膜上皮细胞分离培养方法,为研究病毒、细菌、营养元素以及毒物等与子宫相互作用机理提供研究平台和模型。试验采用4~6月龄胎牛子宫为研究对象,对子宫组织进行类器官培养,比较不同胎龄胎牛子宫类器官培养成功率,然后从类器官出芽组织分离获得原代子宫内膜上皮细胞,并与组织块分离法培养的细胞形态学、纯化培养、生长速度等方面进行比较,采用免疫细胞化学法对获得的子宫内膜上皮细胞进行角蛋白表达的鉴定。结果表明用子宫内膜组织进行类器官培养,4月龄胎牛子宫类器官的出芽率显著高于5、6月龄(P<0.05)。通过类器官出芽进行原代子宫内膜上皮细胞的培养,相较于传统的组织块分离法,更易获得子宫内膜上皮细胞,且获得的细胞形态良好,纯度在95%以上,能够稳定传至8代。研究表明,低月龄的胎牛子宫组织块更易于获得类器官;通过类器官出芽组织获得子宫内膜上皮细胞是一种较为简便、高效的方法,且形态均一、活性好,传代时间更长。

This study was to establish a method for isolation and culture of uterine organs and endometrial epithelial cells of fetal bovine, so as to form a research platform and model foundation for studying the interactional mechanism between viruses, bacteria, nutrients and poisons and uterine. In the experiment, fetal bovine uteruses of 4-6 months of age were used to culture uterine organoid, and the success rates of uterine organoids from different fetal bovine age groups were compared. Primary endometrial epithelial cells were isolated from the organoid budding tissue. Then, the cultured cells were compared with the cells cultured by tissue mass separation in terms of morphology, purification culture, and growth rate. The keratin expression in the obtained endometrial epithelial cells was identified by immunocytochemistry. The results showed that the budding rate of uterine organoids of 4 months of age was higher than that of 5 and 6 the uterine organoids of months of age(P<0.05). Compared with the traditional tissue mass separation method, the primary endometrial epithelial cells cultured from the buddings of organoids were more easily obtained, and the morphology of the cells was better, with a purity of over 95%, which could be stably transmitted to the 8;generation. Therefore, it was easier to obtain the similar organoids using 4-month-old fetal bovine uterine tissue. This method was simple and efficient in obtaining endometrial epithelial cells with uniform morphology, good activity and longer passage time from organoid budding tissue.