山羊胎儿睾丸细胞体外分离培养及雄性生殖系干细胞的生长行为

农业生物技术学报Journal of Agricultural Biotechnology 2004,12(6):648~652

*基金项目：国家自然科学基金项目（No.39970363）、教育部重大项目(No.03160)和国家重点基础研究发展规划（973）项目（No.G1999054300）资助。董武子：（1969年生），男，讲师，博士研究生。E-mail:.**通讯作者。Author for correspondence.E-mail:.收稿日期：2003-12-25接受日期：2004-03-16

·研究论文

·山羊胎儿睾丸细胞体外分离培养及雄性生殖系干细胞的生长行为*

董武子1

华进联1

庄淑珍1沈文正1,2窦忠英1**

（1.西北农林科技大学国家干细胞工程技术研究中心陕西分中心，杨凌712100；

2.杨凌职业技术学院，杨凌712100）

摘要：采用4种不同的处理方法消化山羊胎儿睾丸组织，其中0.1%的胶原酶玉处理15min 后，用0.25%胰酶处理5min ，经3次洗涤，睾丸细胞分散较好。用不同的培养体系体外培养，结果显示：原代培养山羊胎儿睾丸细胞培养120h 左右，获得桑椹状雄性生殖系干细胞(mGSCs)集落和单层支持细胞，mGSCs 集落呈半悬浮式隆突生长，mGSCs 集落和支持细胞分区明显；传代培养显示，在小鼠成纤维细胞饲养层上，mGSCs 集落和mGSC 单细胞被同质化程度较显著，而含有支持细胞的mGSC 单细胞和mGSCs 集落被同质化程度较弱；传代mGSCs 集落和支持细胞共培养体系中，mGSCs 集落正常生长，而且集落中细胞间隙紧密，mGSCs 分化较慢。

关键词：山羊胎儿睾丸；雄性生殖系干细胞；支持细胞；体外培养；生长行为

Separation and

Culture of the Goat Fetal Testicular Cells and the Behavior of Male Germ-line Stem Cells(mGSCs)

DONG Wu-Zi 1HUA Jin-Lian 1ZHUANG Shu-Zhen 1SHEN Wen-Zheng 1,2DOU Zhong-Ying 1**

(1.Shaanxi Province Branch Center of National Stem Cell Engineering &Technology Center,

Northwest Sci-tech University of Agriculture and Forestry,Yangling 712100,China;

2.Yangling Vocational and Technical College,Yangling 712100,

China)

The seminiferous tubules tissues from goat fetus were treated with 4methods.The treatment with 0.1%colleagenase

for 15min and then with 0.25%trypsin for 5min showed ideal cell dispersal degree after centrifugation and washing.The

cul-ture of the cells separated with different medium system showed that mulberry-shaped mGSCs clusters and a single layer of Sertoli cells appeared as original generation in 120h,the mGSCs clusters developed half-suspendedly and distributed in different locations with the Sertoli cells in the plate;The cells in the margin area of incompletely digested mGSCs clusters and the single cells from them were obviously homogenized after culture for 5days in fiber cell feeding layer from mice fetus,while same-treated mGSCs clusters co-cultured with Sertoli cells did not displayed clear homogenization in marginal cells of the clusters in the same culture system;And the first generation of mGSCs clusters co-cultured with Sertoli cells formed the same morphologic properties with the original genera-tion of clusters,cells in clusters were tight and mGSCs divided slower than one in MEF feeding

layer.

of goat fetus;male germ-line stem cells;Sertoli cells;

culture;development behavior

雄性生殖系干细胞（male germ-line stem cells,mGSCs ）是包括性分化后到精子发生过程中，具有自我增殖和分化潜能的细胞总称；不同于精原干细胞（spermatogonial stem cells,SSCs ），精原干细胞仅指未分化的A 型精原细胞[1,2]。应用mGSCs 的增殖分化特性，通过mGSCs 的移植来治疗雄性不育[3]；将基因导入mGSCs 研究基因功能和mGSCs 的行为[4,5]；另外，mGSCs 也是研究生殖细胞减数分裂的极好材料。大量研究集中在mGSCs 具有与其它干细胞有相同的多能性特性并表达特异的细胞表面标