山羊睾丸支持细胞分离培养及其氧化应激效果研究

Isolation, culture and oxidative stress of goat Sertoli cells

关键词:  山羊  支持细胞  分离和培养  氧化应激

Key words:  goat  Sertoli cells  separation and culture  oxidative stress

摘要:

为探究在体外培养过程中山羊睾丸支持细胞（GSCs）存在的氧化应激问题，采用2步酶消化法分离和纯化GSCs，通过形态学观察、MTT法、油红染色和流式细胞鉴定细胞活度和纯度，再用不同浓度的H₂O₂处理GSCs，研究GSCs的活性、丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性及其DNA指数（DI）的变化。结果表明：GSCs形态为多角形状；在培养至第5天时GSCs活性最强；油红O染色鉴定多数细胞胞质含有红色的脂滴；GATA4阳性细胞率达到88.27±3.29%；添加50 μmol/L H₂O₂组细胞存活率与对照组相比差异不显著（P>0.05），其它各组均显著低于对照组（P<0.05）；添加50 μmol/L H₂O₂组MDA含量和SOD的活性均与对照组无显著性差异（P>0.05），而其他各组均与对照组有显著差异（P<0.05）；细胞DNA倍体检测发现添加50 μmol/L H₂O₂时，细胞DI为1.08±0.01，未出现异倍体；其他各组细胞DI分别为1.15±0.02、1.20±0.03、1.26±0.03和1.32±0.01，均有异倍体产生。本研究分离纯化了GSCs，并进行培养和鉴定，200 μmol/L H₂O₂处理GSCs后，SOD活性显著降低，MDA含量显著增加，出现明显的异倍体，200 μmol/L H₂O₂是建立GSC氧化应激模型的适宜浓度，以此为后期建立GSC氧化应激模型提供研究基础。

Abstract:

To study the oxidative stress of goat Sertoli cells (GSCs) during culturing in vitro, a two-step enzyme digestions were used to isolate the testicular cells,then the cells were purified by Tris-HCl treatment. The activity and cell DNA index (DI) were then analyzed. The results showed:The GSCs were polygonal. The cultured GSCs were the most active at fifth day. Most cells contained red lipid droplets in cytoplasm by Oil Red O staining. The GATA4 positive cell rate was 88.27±3.29%. No significant differences in the cell survival rate were observed between the control group and 50 μ mol/L H₂O₂ group (P>0.05). However, the cell survival rates in the other groups were significantly lower than that in the control group (P<0.05). No significant differences in MDA content and SOD activity were observed between the control group and 50 μ mol/L H₂O₂ group (P>0.05). The other groups were significantly different from the control group (P<0.05). The DI in 50 μ mol/L H₂O₂ group was 1.08±0.01, the GSCs in the group did not appear the heteroploid by the cell DNA ploidy test; The DI in other groups was respectively 1.15±0.02, 1.20±0.03, 1.26±0.03, 1.32±0.01 and the cells were all produced the heteroploid. In conclusion, the GSCs were separated, purified, cultivated and identified in the study. After treated with 200 μ mol/L H₂O₂, the SOD activity of the GSCs significantly decreased, the MDA content significantly increased, and there were significant allopolyploids. The optimized concentration was 200 μ mol/L H₂O₂. This study provided basis for the establishment of GSC oxidative stress model.