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目的评价锌指蛋白146(RNF146)对化疗药吡柔比星引起的GC-1小鼠睾丸精原细胞DNA损伤的保护作用。方法采用1.0μg/m L吡柔比星处理的方法复制DNA损伤的小鼠GC-1精原细胞模型。构建小鼠rnf146慢病毒过表达载体p CDHrnf146并包装rnf146过表达慢病毒,感染GC-1细胞上调rnf146的表达,Western blot验证过表达效率。评价RNF146对吡柔比星引起的GC-1细胞增殖抑制、凋亡增多及DNA损伤的保护作用。结果成功构建了p CDH-rnf146重组质粒并包装具有良好感染效率的RNF146过表达慢病毒。溴脱氧尿苷(Brd U)掺入标记显示RNF146能够减轻吡柔比星引起的GC-1增殖抑制。原位末端转移酶标记技术(TUNEL)提示RNF146能够抑制吡柔比星诱导的GC-1细胞凋亡。彗星实验显示RNF146过表达能够有效保护吡柔比星作用下GC-1细胞DNA的完整性。结论 RNF146对化疗药吡柔比星引起的小鼠睾丸精原细胞损伤具有保护作用。
Objective To evaluate the protective effect of zinc finger protein 146 (RNF146) on the DNA damage of GC-1 mouse testicular spermatogonia induced by pirarubicin. Methods A DNA-damaged mouse GC-1 spermatogonia model was replicated using 1.0 μg / mL of pirarubicin. The mouse rnf146 lentivirus overexpression vector pCDHrnf146 was constructed and packaged with rnf146 overexpression lentivirus. The infection of GC-1 cells upregulated the expression of rnf146, and the efficiency of overexpression was verified by Western blot. To evaluate the protective effects of RNF146 on pirarubicin-induced GC-1 cell proliferation, apoptosis and DNA damage. Results The pCDH-rnf146 recombinant plasmid was successfully constructed and packaged with RNF146 overexpression lentivirus with good infection efficiency. Incorporation of bromodeoxyuridine (BrdU) showed that RNF146 attenuated the inhibition of GC-1 proliferation induced by pirarubicin. TUNEL suggested that RNF146 could inhibit pirarubicin-induced apoptosis of GC-1 cells. Comet assay showed that RNF146 overexpression can effectively protect the DNA integrity of GC-1 cells under the action of pirarubicin. Conclusion RNF146 has a protective effect on the injury of mouse testicular spermatogonia induced by pirarubicin.