小鼠睾丸支持细胞体外培养特性

生物工程学报Chin J Biotech2009, May 25; 25(5): 745-753 Chinese Journal of Biotechnology ISSN 1000-3061

cjb@© 2009 Institute of Microbiology, CAS & CSM, All rights reserved

小鼠睾丸支持细胞体外培养特性

师冰洋1, 张淑香1, 郭美锦1, 王永红1, 张嗣良1, 史小林2

1 华东理工大学生物反应器工程国家重点实验室, 上海 200237

2 首都医科大学生殖医学中心, 北京 100069

摘要:支持细胞是睾丸的重要组成部分, 其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持

细胞, 并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、

生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的

研究, 发现支持细胞的贴壁时间主要集中在接种后2~4 h; 当培养液中氨根离子浓度高于 2.3 mmol/L, 渗透压高于

326 mosm/kg, pH≤6.8时支持细胞生长进入衰亡期; 在氨基酸代谢方面, 发现培养过程中丙氨酸和谷氨酸浓度迅速增加,

缬氨酸、亮氨酸和异亮氨酸浓度略有降低, 丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的

过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养

及工艺优化奠定了基础。

关键词:支持细胞, 分离与鉴定, 体外培养, 代谢特性

Metabolic characterization of rat sertoli cell in vitro culture

Bingyang Shi1, Shuxiang Zhang1, Meijin Guo1, Yonghong Wang1, Siliang Zhang1, and Xiaolin Shi2

1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

2 Reproductive Medical Center of Capital Medical University, Beijing 100069, China

Abstract: Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat’s testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated in

vitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine,

amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2−4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressure

above 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors for

SCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.

Received:December 9, 2008; Accepted:February 19, 2009

Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2007AA02Z216), Shanghai

Biotechnology Industrialized Platform (No. 07DZ22914), National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204). Corresponding author: Shuxiang Zhang. E-mail: zhangsx1976@

Meijing Guo. E-mail: guo_mj@

国家高技术研究发展计划(863计划)(No. 2007AA02Z216), 上海市生物技术产业化平台(No. 07DZ22914), 国家重点实验室专项经费

(No. 2060204)资助。