大鼠睾丸生精细胞共培养系统的建立

Establishment of Co-culture System of Rat Spermatogenic Cells

目的建立生精细胞体外分化（如减数分裂、精子变态）培养体系,为研究精子发生中各种调控因素的作用提供一个良好的细胞模型。方法取20-22日龄的SD雄性大鼠睾丸,放在预冷的Hanks液中洗2次,用1 mg/mL胶原酶32℃消化15-20 min。然后将睾丸剪碎,重复用1 mg/mL胶原酶消化5-10 min。细胞用含10%胎牛血清及各种营养因子的HamF12/DMEM培养液悬浮,按约2×10^6/cm^2密度分别接种于双室培养槽（双室培养）或放有盖玻片的6孔板（单室培养）中,在32℃、95%空气、5%CO2条件下培养。每日在相差显微镜下观察共培养支持细胞/生精细胞的生长状态,定期进行苏木精-伊红染色、BrdU染色。结果在双室培养2周后,部分生精细胞可见鞭毛出现,培养4周后,培养体系中仍有一定数量的生精细胞附着在支持细胞上,部分生精细胞上可见鞭毛。单室培养的生精细胞在培养第4-5天开始出现明显脱落,培养1周后大多数生精细胞发生脱落死亡,生精细胞上未见鞭毛出现,但在培养体系中可见次级精母细胞及精子细胞。结论应用双室培养,生精细胞可存活达1月之久,而且部分生精细胞尾部出现鞭毛,从形态上发生了减数分裂及精子变态过程。

Objective To establish a long term spermatogenic cells co-culture system to allow spermatogenic cells to undergo differentiation, such as meiotic process, spermiogenesis. Methods The seminiferous tubules samples from rats aged 20- 22 days were digested with 1 mg/mL collagenase at 32 ℃ for 15- 20 rain, then cut into small fragments. Tubular fragments were digested with collagenase again for 5 10 rain,then gently resuspended in HamF12/DMEM supplemented with various nutri tion factors and 10% fetal calf serum, and seeded at about 2 × 10^5; eells/cm^2 into bicameral chambers or culture plates covered with slides. Co-culture was carried out at 32 ℃ in a water-saturated atmosphere of 95% air and 5% CO2. The growth and morphology of co-cuhured ceils were monitored daily under a contrast phase microscope, and idemified by HE staining, grdU staining regularly. Results In bicameral cuhure system, flagella were seen emerging from one end of some spermatogenic cells after two week co-culture. After four week co culture a certain number of spermalogenie cells still attached on the surface of sertoli cells, while some of them had a flagellum at one end. In unicameral culture system, spermatogenic cells began obviously to fall off the surface of sertoli cells after 4-5 day culture, and there were only a few spermatogenic cells attaching the surface of sertoli cells after one-week co-culture. But the second spermatocytes and spermatids were observed except for flagella in this unicameral coculture system. Conclusion The spermatogenic cells in bicameral culture system survived longer and differentiated easier than those in unicameral culture system. Morphologically, meiotic process and spermiogenesis occurred in the co-culture system.