Stra8过表达精原细胞的细胞周期同步化方法的建立

Establishment of cycle synchronization for spermatogonia with Stra8 overexpression

目的建立Stra8过表达精原细胞(Stra8-GC1)的细胞周期同步化方法,并应用流式细胞仪进行检测。方法 G1期同步化:应用血清剥夺方式分别培养Stra8-GC1 24h、36h、48h、60h、72h、84h和96h,收集细胞并应用流式细胞仪检测G1期细胞百分比;S期同步化:应用含有胸苷培养液培养Stra8-GC1 14~16h,更换正常培养基培养9h,再次加入胸苷培养14~16h,最后更换正常培养基分别培养0、3h,6h和9h,收集细胞并应用流式细胞仪检测S期细胞百分比;M期同步化:应用含有诺考达唑培养液分别培养Stra8-GC1 4h,8h和12h,收集细胞并应用流式细胞仪检测M期细胞百分比。结果血清剥夺处理72h,Stra8-GC1 G1期细胞百分比为60%~70%,显著高于对照组的31.73%(P<0.05);二次胸苷阻滞法处理以及第二次释放3h,Stra8-GC1 S期细胞百分比为60%~70%,显著高于对照组的38.50%(P<0.05);诺考达唑处理8h,Stra8-GC1 M期细胞百分比为90%以上,显著高于对照组的13.85%(P<0.05)。结论成功构建Stra8过表达细胞的细胞周期同步化方法,为后续Stra8的功能研究奠定了基础。

Objective To establish a cell cycle synchronization method for spermatogonia with Stra8 overexpression（Stra8-GC1）. Methods G1 phase synchronization： Stra8-GC1 was cultured in medium with serum deprivation for 24,36,48,60,72,84,96 h, then cells were harvested and the percentage of G1 phase was detected by flow cytometry; S phase synchronization： Stra8-GC1 was successively cultured in medium with thymidine for 14-16 hours, in normal culture medium for 9 hours, in medium within thymidine for 14-16 hours again, finally in normal culture medium for 0, 3, 6 and 9 hours, then cells were harvested and the percentage of S phase was detected by flow cytometry; M phase synchronization： Stra8-GC1 was cultured in medium with nocodazole for 4,8,12 h, then cells were harvested and the percentage of M phase was detected by flow cytometry. Results After the treatment of serum deprivation for 72 h, the percentage of G1 phase cells of stra8-GC1（60-70%） was significantly higher than that of the control group（31.73%, P0.05）. After the treatment of double thymidine block and second release time for 3 h, the percentage of S phase cells of Stra8-GC1（60-70%） was significantly higher than that of the control group（ 38.50%, P0.05）. The percentage of M phase cells of Stra8-GC1 treated with nocodazole for 8 h was more than 90%, which was significantly higher than that of the control group（13.85%, P 0.05）. Conclusion The cell cycle synchronization method of Stra8 over-expression cells was successfully constructed, which laid the foundation for the functional research of Stra8.