Stra8过表达精原细胞的细胞周期同步化方法的建立

摘要

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-GC124h、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-GC114～16h,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-GC14h,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 24,36,48,60,72,84,96h , 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 4,8,12h, then cells were harvested and the percentage of M phase was detected by flow cytometry. Results After the treatment of serum deprivation for 72h, the percentage of G1 phase cells of stra8-GC1 (60～70％) was significantly higher than that of the control group(31.73％, P<0.05). After the treatment of double thymidine block and second release time for 3h, the percentage of S phase cells of Stra8-GC1(60-70％) was significantly higher than that of the control group( 38.50％, P<0.05). The percentage of M phase cells of Stra8-GC1 treated with nocodazole for 8h 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.