上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

二甲磺基乙烷处理生精小管体外培养模型的建立

张磊1,3，张齐好2,3，苏志坚2,3，黄亚东2,3   

  1. 1.广东省生物制品与药物研究所， 广州 510440； 2.暨南大学细胞生物学系， 广州 510632； 3.暨南大学医药生物技术研究开发中心，  广州 510632
  • 出版日期:2015-03-28 发布日期:2015-03-26
  • 通讯作者: 苏志坚, 电子信箱: tjnuszj@jnu.edu.cn; 黄亚东, 电子信箱: tydhuang@jnu.edu.cn。
  • 作者简介:张磊（1982—）， 男， 博士生; 电子信箱: swlxzl@126.com。
  • 基金资助:

    国家自然科学基金（81070477，31000663,31271607）；中央高校基本科研业务费专项资金（21611378）；广东省高等学校珠江学者岗位计划资助项目（2012）

Establishment of culture model of seminiferous tubules treated by ethane dimethanesulfonate in vitro

ZHANG Lei1,3, ZHANG Qi-hao2,3, SU Zhi-jian2,3, HUANG Ya-dong2,3   

  1. 1.Guangdong Provincial Institute of Biological Products and Materia Medica， Guangzhou 510440， China； 2.Department of Cell Biology, Jinan University, Guangzhou 510632, China; 3.Biopharmaceutical Research and Development Center, Jinan University， Guangzhou 510632, China
  • Online:2015-03-28 Published:2015-03-26
  • Supported by:

    National Natural Science Foundation of China, 81070477, 31000663, 31271607； Fundamental Research Funds for the Central Universities of China, 21611378； Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme 2012

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摘要：

目的 建立二甲磺基乙烷（EDS）处理生精小管的体外培养模型。方法 雄性SD大鼠于实验前7 d腹腔注射EDS，分离生精小管进行体外培养，分为基础处理组（加入胰岛素-转铁蛋白-亚硒酸钠培养基添加剂ITS，即ITS组）和促黄体素(LH)处理组（加入ITS和 LH，即ITS+LH组），采用 EdU标记技术检测睾丸间质干细胞增殖情况，放射免疫分析技术及3β-HSD染色检测睾酮分泌及睾丸间质干细胞的分化情况。取雄性成年小鼠睾丸并分离生精小管，分别以不同浓度的EDS处理，其后再进行分组（ITS组和ITS+LH组）处理连续培养，采用放射免疫法检测培养基上清液中睾酮的分泌情况。结果 与ITS组比较，大鼠生精小管ITS+LH组睾丸间质干细胞增殖较为明显。睾酮的分泌呈现与体内睾丸间质细胞发育分化趋势一致；且该模型在LH作用下，睾酮产生量大，持续时间长，表明该模型对LH敏感。1.72 mmol/L EDS可有效杀死小鼠生精小管上的成熟睾丸间质细胞；该处理模型在经过培养后又有睾酮的产生，且ITS+LH组生精小管培养上清液中睾酮含量显著高于ITS组(P<0.01)，表明EDS可以杀死成熟睾丸间质细胞，同样该模型也表现出对LH敏感。结论 雄性大鼠和小鼠生精小管体外培养模型中，睾丸间质干细胞的发育分化过程与体内睾丸间质干细胞的发育分化相似，该模型是一个研究睾丸间质细胞发育分化的合适模型。

关键词: 生精小管, 睾丸间质细胞, 二甲磺基乙烷, 促黄体素

Abstract:

Objective To establish the culture model of seminiferous tubules treated by ethane dimethanesulfonate (EDS) in vitro. Methods Male SD rats were intraperitioneally injected with EDS 7 d before experiment. Seminiferous tubules of rats were removed and cultured in vitro, and divided into the basic treatment group [treated by insulin-transferin-selenium (ITS), i.e. the ITS group] and luteinizing hormone (LH) treatment group (treated by ITS and LH, i.e. the ITS+ LH group). EdU staining was adopted to detect the proliferation of stem Leydig cells. Radioimmunoassay and 3β-HSD staining were employed to detect the secretion of testosterone and differentiation of stem Leydig cells. Seminiferous tubules were isolated from testicles of adult male mouse and treated by EDS of different concentrations. Then seminiferous tubules were treated by ITS (ITS group) and ITS+LH (ITS+ LH group). The secretion of testosterone of culture supernatant was detected by the radioimmunoassay. Results Compared with the ITS group, the proliferation of stem Leydig cells of seminiferous tubules of the ITS+LH group was more significant. The secretion of testosterone was consistent with the development and differentiation of Leydig cells. The production of testosterone of the ITS+LH group was large and the duration was long after being treated by LH, which showed that the culture model of seminiferous tubules was sensitive to LH. EDS of 1.72 mmol/L effectively killed mature Leydig cells of seminiferous tubules. Testosterone was produced after the model was cultured and the testosterone level of culture supernatant of seminiferous tubules of the ITS+LH group was significantly higher than that of the ITS group (P<0.01), which indicated that EDS could kill mature Leydig cells and the model was also sensitive to LH. Conclusion The development and differentiation processes of stem Leydig cells of the male rat and mouse models and the culture model of seminiferous tubules in vitro are similar. The culture model is suitable for studying the development and differentiation of Leydig cells.

Key words: seminiferous tubules, Leydig cells, ethane dimethanesulphonate, luteinizing hormone