中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (31): 5797-5800.doi: 10.3969/j.issn.1673-8225.2010.31.024

• 器官移植动物模型 organ transplantation and animal model • 上一篇    下一篇

一种获得高纯度睾丸支持细胞的分离方法　

麦海星，陈立军，曲  楠，赵  立，黄  晨，王亚林，李学超　　   

  1. 解放军军事医学科学院附属医院泌尿外科，北京市  100071
  • 出版日期:2010-07-30 发布日期:2010-07-30
  • 通讯作者: 陈立军，副教授，硕士生导师，解放军军事医学科学院附属医院泌尿外科，北京市 100071 chenlj517@hotmail.com
  • 作者简介:麦海星★，男，1981年生，广东省阳江市人，汉族，2008年解放军第四军医大学毕业，硕士，医师，主要从事肾脏移植免疫耐受研究。 maimark24@hotmail.com
  • 基金资助:

    陕西省自然科学基金(2005K14-G4(3))资助项目。　

An isolation method to obtain high purity Sertoli cells

Mai Hai-xing, Chen Li-jun, Qu Nan, Zhao Li, Huang Chen, Wang Ya-lin, Li Xue-chao　   

  1. Department of Urology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing  100071, China
  • Online:2010-07-30 Published:2010-07-30
  • Contact: Chen Li-jun, Associate professor, Master’s supervisor, Department of Urology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing 100071, China chenlj517@hotmail.com
  • About author:Mai Hai-xing★, Master, Physician, Department of Urology, Affiliated Hospital of Academy of Military Medical Sciences, Beijing 100071, China maimark24@hotmail.com
  • Supported by:

    the Natural Science Foundation of Shaanxi Province, No. 2005K14-G4(3)*

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摘要：

背景：研究证实睾丸支持细胞具有免疫抑制功能，可以被用于在睾丸以外的部位为移植细胞提供免疫豁免环境。最近的研究还表明支持细胞分泌的多种活性物质对其他细胞有促进作用。但至今其分离纯化方法还不完整，且细胞纯度不太理想。
目的：探讨一种获取高纯度睾丸支持细胞的方法。
方法：迅速分离出生后20 d 雄性SD大鼠睾丸生精小管，采用酶消化法将细胞悬液接种于多聚赖氨酸包被的培养板内培养7 d后，采用Tris-HCL低渗缓冲液处理生精细胞和成纤维细胞，采用FasL免疫组织化学、Hoechst33342复染鉴定睾丸支持细胞的纯度。
结果与结论：支持细胞贴壁性比较强，培养24 h后，大多数细胞开始贴壁，细胞形状呈圆形或椭圆形，培养液中漂浮着较多生精细胞。培养48 h后，胞质逐渐增多，折光性减弱。三四天后支持细胞胞质铺展，细胞间隙变小。4~6 d后胞质完全铺开，细胞相互连接，铺展成膜状单层。此时，质核比为(7~9)∶1，细胞为多边形，其形态和胞质面积不再改变；睾丸支持细胞的纯度为(95.64±2.76)%。结果证明该方法是一种相对简便易行且经济﹑稳定﹑有效的睾丸支持细胞分离方法。

关键词: 分离, 原代培养, FasL, 睾丸支持细胞, 器官移植基础实验

Abstract:

BACKGROUND: Studies demonstrated that Sertoli cells have immunosuppression, which can provide immune privilege for transplanted cells in vitro. Previous researches indicated that Sertoli cells-secreted active compounds have promotive effects on other cells. However, there is not a complete method for Sertoli cells purification.
OBJECTIVE: To discuss a method for isolating Sertoli cells with high purity.
METHODS: The testicular seminiferous tubules were isolated from 20-day old male SD rats, digested by enzyme and were plated onto poly-L-lysine (PLL)-coated tissue culture dishes followed by 7 days culture, and then incubated with Tris-HCL. Purity of Sertoli cells were identified by immunohistochemistry of FasL with a counterstain of Hoechst33342.
RESULTS AND CONCLUSION: Most Sertoli cells began to stick on the flasks with round or oval shapes at 24 hours after culture, and many germ cells could be seen floating on the liquid. The cytoplasty become greater and refraction turned to weakening at 48 hours after culture. After 4-6 days, cells were fully spread and become monolayer. And the mass to charge ratio was (7-9):1, the shape and proportion of the cells had no change. The purity of the Sertoli cells was (95.64±2.76)%. The results demonstrated that this method is a relatively easy, cheap, stable, and efficient for isolating Sertoli cells.

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