绵羊绒毛膜滋养层细胞与子宫内膜腔上皮细胞共培养体系的建立

doi:10.11843/j.issn.0366-6964.2019.03.011

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (3): 562-569.doi: 10.11843/j.issn.0366-6964.2019.03.011

绵羊绒毛膜滋养层细胞与子宫内膜腔上皮细胞共培养体系的建立

张洁¹, 陈大勇², 赵娟¹, 王晓娟¹, 杨惠¹, 刘淑英^1,3*

1. 内蒙古农业大学兽医学院, 呼和浩特 010018;
2. 内蒙古赛诺种羊科技有限公司, 呼和浩特 010018;
3. 农业部动物临床诊疗技术重点实验室, 呼和浩特 010018

收稿日期:2018-07-09 出版日期:2019-03-23 发布日期:2019-03-25
通讯作者: 刘淑英,主要从事生殖内分泌与病毒病理学研究,E-mail:liushuying_imau@126.com
作者简介:张洁(1993-),女,内蒙古包头人,硕士生,主要从事病毒病理学研究,E-mail:zj1060356734@163.com
基金资助:
国家自然科学基金（31760721）；内蒙古草原英才创新团队项目（20151031）；内蒙古科技应用研究项目（2018）

The Establishment of Co-culture System of Sheep Trophoblast Cells and Endometrial Luminal Epithelial Cells

ZHANG Jie¹, CHEN Dayong², ZHAO Juan¹, WANG Xiaojuan¹, YANG Hui¹, LIU Shuying^1,3*

1. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Inner Mongolia Sainuo Sheep Pasture Corporation, Hohhot 010018, China;
3. Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture, Hohhot 010018, China

Received:2018-07-09 Online:2019-03-23 Published:2019-03-25

摘要/Abstract

摘要：

本研究旨在探索绵羊绒毛膜滋养层细胞（sheep trophoblast cells，STCs）和子宫内膜腔上皮细胞（endometrial luminal epithelial cells，ELECs）的共培养条件，为绵羊胎盘绒毛膜滋养层多核细胞的形成提供试验依据。在屠宰场采集妊娠45~60 d的健康绵羊子宫及胎盘组织，用酶消化法体外分离培养STCs和ELECs，并进行细胞免疫荧光鉴定，同时利用姬姆萨氏染色法确定STCs和ELECs最佳的共培养条件；然后转染空质粒pEGFP-C1到ELECs 48 h后，与STCs共培养，并利用激光共聚焦显微镜观察滋养层多核细胞的形态。结果显示：STCs CK-7与ELECs CK-18均呈阳性；当STCs与ELECs细胞数量比值为2：1，共培养48 h，多核细胞数量最多，且与对照组相比差异显著（P<0.05）；CK-7在STCs中表达呈红色荧光，且细胞中有pEGFP-C1标记的绿色荧光，胞核蓝染，3种荧光共表达呈现出了白光。综上表明，本研究建立了STCs和ELECs共培养条件，ELECs参与绵羊滋养层多核细胞的形成。

关键词: 绵羊, 滋养层细胞, 子宫内膜腔上皮细胞, 共培养, 绒毛膜滋养层多核细胞

Abstract:

The study aims to explore the co-culture conditions of sheep trophoblast cells (STCs) and the endometrial luminal epithelial cells (ELECs), and provides experimental basis for chorionic trophoblastic coenocyte formation. Harvesting healthy sheep uterus and placental tissue from 45 to 60 days of gestation at the slaughter house, and the enzyme digestion method has been used to isolate and culture the STCs and ELECs in vitro, and they were identified by cellular immunofluorescence. Simultaneously, the optimal co-culture conditions for STCs and ELECs were determined by Giemsa staining. Then, the empty plasmid pEGFP-C1 was transfected into ELECs for 48 h, and then co-cultured with STCs. The morphology of chorionic trophoblastic coenocyte was observed by confocal laser scanning microscopy. The results showed that STCs CK-7 and ELECs CK-18 were positive.When the ratio of STCs to ELECs cells was 2:1, the number of multinucleated cells reaches the maximum when the co-culture time was 48 h, and it was significantly different from the control group (P<0.05). The results of immunofluorescence showed that CK-7 expressed red fluorescence in STCs, and there were pEGFP-C1 labeled green fluorescence in the cells, and the nucleus was blue-stained, and the 3 fluorescent co-expressions showed white light. This study established the conditions for co-culture of STCs and ELECs, and showed that ELECs were involved in the formation of polynuclear cells in sheep trophoblasts.

Key words: sheep, trophoblast cells, endometrial luminal epithelial cells, co-culture, chorionic trophoblastic coenocyte

中图分类号:

S826.2

张洁, 陈大勇, 赵娟, 王晓娟, 杨惠, 刘淑英. 绵羊绒毛膜滋养层细胞与子宫内膜腔上皮细胞共培养体系的建立[J]. 畜牧兽医学报, 2019, 50(3): 562-569.

ZHANG Jie, CHEN Dayong, ZHAO Juan, WANG Xiaojuan, YANG Hui, LIU Shuying. The Establishment of Co-culture System of Sheep Trophoblast Cells and Endometrial Luminal Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 562-569.

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绵羊绒毛膜滋养层细胞与子宫内膜腔上皮细胞共培养体系的建立

The Establishment of Co-culture System of Sheep Trophoblast Cells and Endometrial Luminal Epithelial Cells

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