大鼠睾丸曲细精管组织支持细胞/生殖细胞长期共培养与精子发生分析

Long-term Culture and Spermatogenesis Analysis of Sertoli Cell/Germ Cell Co-culture System from Seminiferous Tubules of Rat Testis

睾丸体外生殖模型的发展为体外研究睾丸的精子发生分子机制和睾丸毒理学提供了实验工具。很多报道的模型都无法真正地模拟体内复杂的生化分子及功能性相互作用从而导致研究价值有限。该实验拟建立一个体外长期维持睾丸生殖细胞存在,并能持续产生精子细胞的支持细胞/生殖细胞共培养体系。体系中的支持细胞和生殖细胞均由曲细精管组织块迁移到培养皿上,在不添加任何生长因子的情况下维持体外精子发生至圆形精子细胞超过2个月。RT-PCR分析显示,共培养细胞稳定表达cdh1、scp3、tnp2;免疫荧光染色结果显示,CDH1、PLZF、SCP3以及SOX9阳性细胞存在。这些结果例证了体系中同时存在精原干细胞、精母细胞、精子细胞和支持细胞。简单高效的支持细胞/生殖细胞体外共培养体系可用于雄性生殖的分子机制和毒理学研究。

The development of in vitro models of testicular reproductive system may provide important tools for investigating specific mechanisms of spermatogenesis and toxicity in the testis. Although various systems have been reported, their application in reproductive studies has been limited by the poor ability to replicate the complex biochemical, molecular, and functional interactions observed in the testis. In the present study, we evaluated a significantly improved sertoli cell/germ cell co-culture （SGC） system that the proliferation and differentiation of testicle germ cells of the rat could be maintained for long-term in vitro, and spermatids produced ceaselessly. The germ cells and sertoli cells came from pieces of the testis seminiferous tubules of the rat. The co-cultured cells survived in vitro for over two months without extra growthfactor. Furthermore, RT-PCR analysed cells stabilization of cdhl 、 scp3 、 tnp2 expression confirmed the integrity of this co-culture system. Immunocytochemistry displayed the CDHI 、 PLZF、 SCP3、 SOX9 positive cells were existed in system. All the results proved that the SGC system included the spermatogonial stem cell, spermatocyte, round spermatid and sertoli cell. We concluded that this system would provide investigators with a simple and efficient alternative for the assessment of molecular mechanisms associated with both male reproduction development and reproductive toxicity.