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。 |
关键词: 山羊 支持细胞 分离和培养 氧化应激 |
DOI:10.11841/j.issn.1007-4333.2019.02.11 |
投稿时间:2018-04-10 |
基金项目:国家自然科学基金项目(31001019);安徽省自然科学基金项目(1708085MC81) |
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Isolation, culture and oxidative stress of goat Sertoli cells |
WANG Juhua1, LIU Dandan1, LIU Qi1, WANG Jiaming1, DONG Jing1, ZHOU Jie1, XUE Xiuheng2
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(1.College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;2.College of Tea & Food Technology, Anhui Agricultural University, Hefei 230036, China) |
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. |
Key words: goat Sertoli cells separation and culture oxidative stress |