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目的 引进精子单细胞凝胶电泳 (single- cell gel electropherosis,SCGE)实验方法 ,用以检测人类精子 DNA链断裂。方法 载有精子和凝胶的玻片首先在 p H10的弱碱性裂解液中作用 1h,精子核分别用 10 mmol/ L 二硫苏糖醇处理 1h,10 μg/ ml RNA酶处理 4h,2 0 0 μg/ ml蛋白酶 K处理 15 h。精子核用15 μg/ ml的 EB染色 5 min。计数彗星细胞百分率 ,并用本试验室建立的精子 SCGE实验方法观察 H2 O2 在体外条件下对正常精子细胞 DNA的损伤作用。结果 8名受试彗星精子细胞百分率为 2 %～ 38% ,不同个体之间有明显差异。精子细胞在 1.0 mmol/ L H2 O2 的 PBS中孵育 0 .5～ 4h,彗星细胞百分率显著增加 ,并有明显的时间 -效应趋势。精子细胞在 0 .12 5～ 1.0 0 0 mmol/ L H2 O2 的 PBS中孵育 1h,彗星细胞百分率显著增加 ,并有明显的剂量 -效应关系。结论 SCGE可用于检测人类精子细胞 DNA链断裂 ,使用这一实验方法成功地检测出 H2 O2 引起的人类精子 DNA损伤。
Objective To introduce sperm single cell gel electrophoresis (SCGE) method to detect human sperm DNA strand breaks. Methods Slides containing sperm and gel were first treated in weak alkaline lysis buffer of p H10 for 1 h, sperm nuclei were treated with 10 mmol / L dithiothreitol for 1 h and 10 μg / ml RNase for 4 h, 2 0 0 μg / ml Proteinase K for 15 h. Sperm nuclei were stained with 15 μg / ml EB for 5 min. The percentage of comet cells was counted and the damage of H2O2 on the DNA of normal spermatozoa cells in vitro was observed by sperm SCGE assay established in this laboratory. Results The percentage of spermatids in 8 tested comets was 2% ~ 38%. There were significant differences among different individuals. Sperm cells incubated in 1.0 mmol / L H 2 O 2 PBS for 0.5 ~ 4 h, the percentage of comet cells increased significantly, and there is a clear trend of time-effect. Sperm cells were incubated for 1 h in 0.1250-1.00 mmol / L H 2 O 2 PBS, the percentage of comet cells increased significantly, and there was a significant dose-effect relationship. Conclusion SCGE can be used to detect DNA strand breaks in human spermatozoa cells. Using this method, human sperm DNA damage induced by H2O2 can be successfully detected.