小鼠睾丸支持细胞体外培养特性

Metabolic characterization of rat sertoli cell in vitro culture

摘要:

支持细胞是睾丸的重要组成部分, 其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持细胞, 并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究, 发现支持细胞的贴壁时间主要集中在接种后2~4 h; 当培养液中氨根离子浓度高于2.3 mmol/L, 渗透压高于 326 mosm/kg, pH≤6.8时支持细胞生长进入衰亡期; 在氨基酸代谢方面, 发现培养过程中丙氨酸和谷氨酸浓度迅速增加, 缬氨酸、亮氨酸和异亮氨酸浓度略有降低, 丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养及工艺优化奠定了基础。

Abstract:

Sertoli cell (SC) is intrinsic to the testis and provides an appropriate growth environment for the germ cells. It was separated from rat’s testis and identified by hematoxylin and eosin staining(HE) and immunocytochemical reaction, then cultivated in vitro. Culture conditions such as pH, osmotic pressure and metabolic parameters that include consumption rates of glucose, glutamine, amino acids and formation rates of lactic acid, ammonium ion were investigated. It was showed that adhesion process of SCs was accomplished within 2-4 hours after inoculation. It was also observed that the SCs entered into the decline phase when the concentration of ammonium ion and lactic acid were above 2.3 mmol/L and 14 mmol/L, respectively, which caused osmotic pressure above 326 mosm/kg and pH below 6.8 in the medium. As the changes of amino acids during culture were concerned, Glu and Ala accumulated rapidly, while Val, Leu, Ile reduced slightly and at the same time Ser, Arg, and Gly were stable. The restrict factors for SCs grown in static culture might be high osmotic pressure and low pH, which were generated when glutamine and glucose were metabolized into lactic acid. The findings could be fundamental in the process optimization of large scale Sertoli cells in vitro culture.