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目的:探讨大鼠睾丸支持细胞原代培养5-溴-2-脱氧尿苷(BrdU)的最佳标记时间、剂量。方法:大鼠睾丸支持细胞进行体外原代培养;以终浓度分别为5、10、15、20μmol/L的BrdU检测睾丸支持细胞的最佳标记剂量;在细胞培养的12、24、36、48h掺入BrdU,使终浓度为15μmol/L,测定BrdU的最佳标记时间;免疫细胞化学法跟踪观察大鼠睾丸支持细胞在体外培养中的分化发育情况和BrdU标记率。结果:免疫组化检查提示最终浓度为15μmol/L和孵育36hBrdU标记率均>90%,并且连续传3代均可检测到。结论:终浓度15μmol/L及孵育36h为BrdU标记睾丸支持细胞的最佳剂量及时间,表明BrdU标记可用于睾丸支持细胞体内后的存活、生长的动态观察。
Objective: To investigate the optimal labeling time and dose of 5-bromo-2-deoxyuridine (BrdU) in primary cultured rat sertoli cells. METHODS: The rat testicular sertoli cells were primarily cultured in vitro. The optimal labeled dose of BrdU for 5, 10, 15, 20μmol / L, respectively, was determined. At 12, 24, 36 and 48h BrdU was added to make the final concentration of 15μmol / L, and the optimal labeling time of BrdU was determined. The differentiation and development of BrdU labeled cells and the rate of BrdU labeling in vitro were observed by immunocytochemistry. Results: Immunohistochemistry showed that the final concentration of 15μmol / L and 36h BrdU labeling rate were> 90%, and three consecutive generations were detected. CONCLUSION: The best dose and time of BrdU labeled testicular sertoli cells at a final concentration of 15μmol / L and 36h incubation indicate that the BrdU labeling can be used to monitor the survival and growth of testicular sertoli cells.