大鼠生精细胞体外培养条件的研究

目的:寻找一个最适合生精细胞体外培养的培养体系,为进一步培养人类生精细胞提供技术基础.方法:用单一酶-研磨-Percoll法制备15 d龄雄性大鼠生精细胞混悬液用于体外培养.①设计两种不同的培养条件:DMEM/F12和改良人类输卵管液(HTF)作为基础培养液,分为两组.每组根据添加不同浓度的激素分为6小组.添加的睾酮(T)和FSH浓度分别为:对照组H0(T 1 μmol/L,FSH 0 IU/L)、H1组(T 1 μmol/L,FSH 10 IU/L)、 H2组(T 1 μmol/L,FSH 25 IU/L)、H3组(T 1 μmol/L,FSH 50 IU/L)、H4组(T 1 μmol/L,FSH 100 IU/L)、H5组(T 0 μmol/L,FSH 50 IU/L). ②通过BRDU标记和流式细胞仪检测所有实验的生精细胞倍体的变化来判断其发育,进一步确认本研究使用培养条件的可靠性.结果:在生精细胞与支持细胞共培养过程中以改良人类输卵管液作为基础培养液,添加T 1 μmol/L ,FSH 50 IU/L或100 IU/L都适宜生精细胞体外培养;另通过BRDU标记及流式细胞仪检测均表明,在本研究条件下体外培养生精细胞能从初级精母细胞阶段分化到精子细胞阶段.结论:改良人类输卵管液作为基础培养液,添加浓度为50 IU/L或100 IU/L的FSH及T 1 μmol/L,最适合生精细胞的体外成熟培养.

A STUDY ON IN-VITRO CULTURE OF MOUSE SPERMATOGONIA

Objective: To find a suitable culture system for spermatocyte in vitro, and give technical support to human spermatocyte culture. Methods: 15-day mouse spermatocytes suspended mixture liquid was made as in-vitro culture media with the method of single enzyme-rubbing-percoll method. The first part: to design two different culture conditions:Group One, the DEME/F12, is a basic culture medium added with some other element; another group, the improved human oviduct liquid, is a basic culture medium added with some other element. The second part: the improved human oviduct liquid is a basic culture medium added with different dosages of FSH. Four groups were designed as T0,T1,T2 and T3 and the FSH dosages are: 0 IU/L,10 IU/L,25 IU/L,50 IU/L and 100 IU/L. The third part: the chromosomal ploidy changes of all cultured spermatogenic cells by marking with BRDU and by the examination with Flow Cytometer System were used as the method to estimate the development of the spermatogenic cells, and then affirm the reliability of the culture condition. Result: The improved human oviduct liquid was preferable to DEME/F12 in spermatogonia in-vitro culture when co-cultuinge the spermatocytes and sertoli cells. Moreover, it has significant influence when adding FSH to spermatocytes in-vitro maturation, and it is the most suitable dosage for the growth. The result of the experiment by BRDU marker and examination with Flow Cytometer System shows that the primary speratocytes can differentiate to sperm cells by in-vitro culture in the study. Conclusion: Improved human oviduct liquid as basic culture medium, added with 50 IU/L FSH, is suitable for spermatocyte maturation in vitro.