改良消化富集法提高精原干细胞富集效率的实验研究  

A modified method for improving the enrichment efficiency of spermatogonial stem cell

作　　者：胡卫 张茨[2] 廖文彪[2] 吴永超[2] 申复进[2] 

机构地区：[1]大冶市中医院泌尿外科,大冶435100 [2]武汉大学人民医院泌尿外科

出　　处：《中国男科学杂志》2009年第6期11-13,共3页Chinese Journal of Andrology

基　　金：国家自然科学基金资助项目(30400160)

摘　　要：目的建立一种改良的小鼠精原干细胞消化富集方法，为精原干细胞体外长期培养和移植奠定基础。方法收集t20个出生4—6dBALB／c新生小鼠的睾丸，平均分为对照组和实验组，分别采用传统的两步消化法（对照组）和改良消化法（实验组）分离消化获得细胞悬液，并种植到铺有明胶的培养皿中，于种植后1h、5h、24h通过差速贴壁法去除体细胞，获得富集的精原干细胞。以Thy．1作为精原干细胞的表面标志，分别在3次差速贴壁前、后采用流式细胞仪检测Thy．1阳性细胞的比例，比较两组精原干细胞的富集效率。结果实验组和对照组消化所获得的细胞数分别为（3．4±0．5）×10^5／睾丸和（3．6±0．3）×10^5／睾丸，无统计学差异（P〉0．05）。然而，实验组细胞贴壁具有良好的活性，初次贴壁仅需40-50min，而对照组细胞需要4-5h甚至过夜培养才能贴壁。3次差速贴壁后，实验组Thy．1阳性细胞占睾丸细胞的比例为（56．3±4．7）％，而对照组仅为（32．6±4．2）％，差异具有统计学意义（P〈0．01）。结论改良消化富集法能明显提高睾丸细胞活性和富集效率，可获得大量高度纯化的精原干细胞，为精原干细胞培养和移植奠定基础。Objective To establish a modified method for the enrichment of mouse spermatogonial stem cells（SSCs）. Methods Total of 120 testes from 4-6 days neonatal mice were collected and randomly divided into two groups such as control group digested with a two-step enzymatic digestion method and experiment group with modified digestion method. Collected cells were plated on culture dish precoated with.0.2% （w/v） gelatinculture The germ cells were purified and enriched by differential adherence selections at 1 h, 5 h and 24h after the cells were seeded. Thy-1 as a surface marker of SSCs was detected by flow cytometry to assess the enrichment efficiency of spermatogonial stem cells. Results The number of cells collected from modified method and traditional digestion method was （3.4±0.5）×10^5 and （3.6±0.3）×10^5 respectively,no significant difference was found （P〉0.05, n=15）. However, collected cells from experiment group showed better activity. It took only 40,450 min for them to stick to the bottom of culture dish, whereas the cells from control group need 4-5h or even 12h to stick the plate after cells were seeded..After three differential adherence selections, the proportions of Thy-1 （＋） cells in experiment group were significantly higher than that in control group （P〈0.01, n=15）. Conclusion The purification and enrichment efficiency of spermatogonial stem cells can be improved by the modified method, which might provide some clues for SSC culture and transplantation.

关 键 词：精原干细胞 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]