绒山羊附睾上皮细胞培养方法的建立及其生长特性

doi:10.11843/j.issn.0366-6964.2017.07.005

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (7): 1212-1220.doi: 10.11843/j.issn.0366-6964.2017.07.005

绒山羊附睾上皮细胞培养方法的建立及其生长特性

范晓梅^1,2, 张通¹, 栗瑞兰¹, 边小娜¹, 张家新^1*

1. 内蒙古农业大学动物科学学院内蒙古自治区动物遗传育种与繁殖重点实验室, 呼和浩特 010018;
2. 内蒙古医科大学基础医学院, 呼和浩特 010110

收稿日期:2017-01-05 出版日期:2017-07-23 发布日期:2017-07-20
通讯作者: 张家新,教授,博士生导师,主要从事动物生殖生物学与繁殖技术研究,E-mail:zjxcau@163.com
作者简介:范晓梅(1980-),女,内蒙古呼和浩特人,副教授,博士生,主要从事动物生理学和生殖内分泌研究,E-mail:1997-lxh@163.com
基金资助:
国家自然科学基金（31460598）；海南省重大科技计划（ZDKJ2016017-02）

Establishment of Culture Method and Growth Characteristics for Epididymal Epithelial Cells from Cashmere Goats

FAN Xiao-mei^1,2, ZHANG Tong¹, LI Rui-lan¹, BIAN Xiao-na¹, ZHANG Jia-xin^1*

1. Key Laboratory of Animal Genetics, Breeding and Reproduction of Inner Mongolia, College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Basic Medical College, Inner Mongolia Medical University, Hohhot 010110, China

Received:2017-01-05 Online:2017-07-23 Published:2017-07-20

摘要/Abstract

摘要：

本研究旨在建立一种简单易行、获取细胞纯度高的绒山羊附睾上皮细胞体外培养体系。10~12月龄绒山羊附睾头部分别采用酶消化法和改良组织块消化法进行原代培养，利用免疫细胞化学染色法和免疫荧光化学法对细胞进行鉴定，同时利用流式细胞仪和CCK法分别检测细胞纯度和增殖情况，在电镜下观察细胞超微结构。结果表明，两种培养方法所获的细胞均呈岛屿状克隆生长和较好的增殖能力，具有活跃的分泌和代谢功能；上皮细胞特异性的角蛋白18和附睾特异性的谷胱甘肽过氧化物酶5（GPx5）的表达以及流式细胞仪结果显示，两种方法所获细胞均为纯度较高的附睾上皮细胞，但改良组织块消化法获得原代细胞需要的时间长，酶消化法获得原代细胞时间短，获得的细胞量大。本研究结果为进一步研究绒山羊附睾上皮功能提供了良好的基础。

关键词: 细胞培养, 附睾上皮细胞, 酶消化法, 改良组织块消化法

Abstract:

The aim of this study was to establish a simple and feasible in vitro culture system for high purity epididymal epithelial cells from cashmere goat. The primary cells were obtained from epididymal caput of 10-12 months cashmere goat using enzymatic dispersion and improved tissue-piece digestion method. The cells were identified by immunocytochemistry and immunofluorescence methods. Cell purity and proliferation were also investigated respectively by flow cytometry and CCK method. Ultrastructures of epididymal epithelial cells were observed under transmission electron microscope. The results showed that the cells obtained by both methods were in island-like clonal growth and good proliferation ability, and possessed active secretory and metabolic function. The expression of epithelial special cytokeratin 18 and epididymal special glutathione peroxidase 5(GPX5) and the result of flow cytometry showed that both methods could obtain high purity epididymal epithelial cells. The primary cells obtained by improved tissue-piece digestion method took a long culture time, compared to that the primary cells obtained by enzymatic dispersion method which took a short culture time with more cells. These results provided a good foundation for further study on the function of epididymal epithelial cells of cashmere goat.

Key words: cell culture, epididymal epithelial cells, enzymatic dispersion method, improved tissue-piece digestion method

中图分类号:

S827
Q24

范晓梅, 张通, 栗瑞兰, 边小娜, 张家新. 绒山羊附睾上皮细胞培养方法的建立及其生长特性[J]. 畜牧兽医学报, 2017, 48(7): 1212-1220.

FAN Xiao-mei, ZHANG Tong, LI Rui-lan, BIAN Xiao-na, ZHANG Jia-xin. Establishment of Culture Method and Growth Characteristics for Epididymal Epithelial Cells from Cashmere Goats[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(7): 1212-1220.

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绒山羊附睾上皮细胞培养方法的建立及其生长特性

Establishment of Culture Method and Growth Characteristics for Epididymal Epithelial Cells from Cashmere Goats

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