BRL饲细胞及血清浓度对体外小鼠精原干细胞的影响

The effects of BRL feeders and serum concentration on mouse spermatogonial stem cells in vitro

分别以大鼠肝细胞(BRL)或小鼠胚胎成纤维细胞(STO)为饲养层,采用含5%、10%及20%胎牛血清的培养液,培养6日龄小鼠生精上皮细胞,研究饲养层和血清浓度对精原干细胞生物学行为的影响。结果表明:在培养的第1周内,2个及4个连成串的精原细胞合胞体数量明显增加,同时大多数精原细胞逐渐退化。培养1周后,大多数精原细胞经短暂的存活及分裂活动后退化消失,培养体系中仅保留下少量单个及由细胞间桥相连的双个及4个成串或成团的A型精原细胞。这些细胞在随后的培养过程中,不表现明显的分裂活动,呈圆球形,表达碱性磷酸酶。不同条件培养体系中的精原细胞及精原干细胞的生物学行为无明显不同,培养20～30d时均有少量精原干细胞存活。这说明BRL细胞能用作饲养层促进精原干细胞存活,但对其更新性增殖没有明显作用;培养液中高浓度胎牛血清对精原干细胞存活和增殖无明显影响,但能促进睾丸体细胞增殖。

The seminiferous epithelial cells dissociated from 6 days postpartum mice were cultured on buffalo rat liver (BRL) cell feeders or on STO feeders with fetal calf serum at different concentrations, respectively, to study the effects of BRL feeders and serum concentration on culture of mouse spermatogonial stem cells. During the first week of culture, the spermatogonial syncytia of 2-and 4-cell increased obviously. At the same time, most spermatogonia degenerated gradually. After a week of culture, only some single spermatogonia, and paired, aligned or clustered spermatogonial syncytia maintained. They showed no obvious proliferation or differentiation in subsequent culture. These cells were spherical and expressed alkaline phosphatase activity. They were spermatogonial stem cells and transient spermatogonia according to the biological characteristics of spermatogonial stem cells. Under different culture conditions mentioned above, the biological behaviors of spermatogonia and spermatogonial stem cells showed no obvious difference and only small number of spermatogonial stem cells remained when they were cultured for 20~30 days. In conclusion, BRL cells can be used as feeders to promote survival but not renewal of spermatogonial stem cells. Fetal culf serum at high concentration have no obvious effects on biological behaviors of spermatogonia and spermatogonial stem cells and can only stimulate proliferation of testicular somatic cells.