绵羊多核绒毛膜滋养层细胞的分离培养与鉴定  

Isolation,culture and identification of sheep multinucleated chorionic trophoblast cells

作　　者：王专家 张宇飞[1] 刘淑英[1,2] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业部动物临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《细胞与分子免疫学杂志》2014年第1期82-86,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：教育部博士点基金(20111515110008);国家自然科学基金(31160493);内蒙古应用技术研究与开发项目(20111803)

摘　　要：目的探索绵羊多核绒毛膜滋养层细胞的分离和培养方法,并进行鉴定。方法采用胰蛋白酶和胶原酶两步消化法分离培养滋养层细胞,倒置相差显微镜观察滋养层细胞的形态学特点;应用常规HE染色和免疫组织化学染色,透射电镜技术进行绵羊多核绒毛膜滋养层细胞鉴定。结果倒置相差显微镜下,滋养层细胞为双核及多核细胞,细胞形态为上皮样,呈片状铺展生长;绵羊胎盘子叶与培养的滋养层细胞爬片的细胞角蛋白免疫组织化学染色均显示多核滋养层细胞胞质为棕色阳性信号,透射电镜可见滋养层细胞表面微绒毛发达,胞质内有较多膜包小泡及丰富的微丝和脂滴。结论建立了胰蛋白酶和胶原酶两步消化法分离培养绵羊多核绒毛膜滋养层细胞的方法,获得了较高纯度的具有生物学活性的多核绵羊绒毛膜滋养层细胞。Objective To develop a set of methods for isolating and culturing sheep multinucleated chorionic trophoblast cells and identify them. Methods Trophoblast cells were isolated and cultured by two-step digestion of trypsin and collagenase, and observed under an inverted phase-contrast microscope. They were further identified through conventional HE staining, immunohistochemical staining and transmission electron microscopy. Results Under an inverted phase-contrast microscope, the obtained trophoblast cells exhibited epithelioid and sheet-like spreading growth with binuclear or multinuclear. The multinucleated trophoblast cytoplasm from the sheep placental cotyledons and the slides of the trophoblast cells were all stained brown and exhibited positive reactivity by immunohistochemical staining with cytokeratin antibodies. Also abundance of microvUli on surface of cells, together with intracytoplasmic vacuoles, microfilament and lipid droplets were observed under a transmission electron microscope. Conclusion Two-step digestion of trypsin and collagenase has been established for the isolation and cultivation of sheep multinucleated trophoblast cells, and using it, we obtained sheep multinucleated chorionic trophoblast cells with a high purity and biological activity.

关 键 词：多核细胞 细胞培养 滋养层 鉴定 

分 类 号：R392-33[医药卫生—免疫学] Q813.11[医药卫生—基础医学]