三种GC-1细胞DNA损伤模型的建立及比较分析

Establishment and comparative analysis of three DNA damage models of GC-1 cells

目的建立3种小鼠睾丸精原细胞株GC-1细胞DNA损伤模型并分析其异同。方法分别采用UVB辐照、D-半乳糖(D-Galactose,D-Gal)、博来霉素(bleomycin,BLM)刺激GC-1细胞不同时间,Western blot和免疫荧光法检测γ-H2AX表达及定位;免疫荧光法检测8-OHdG表达及定位;Western blot法检测p-p53和p21表达水平。结果UVB辐照和D-Gal处理后,γ-H2AX蛋白表达分别在4 h和6 h达高峰BLM刺激后,γ-H2AX蛋白表达逐渐上升且BLM浓度越高,其达到高峰时间越短。UVB辐照和BLM刺激后,胞核胞质内均有8-OHdG表达,且时间越长,核内表达越多;D-Gal处理后,8-OHdG主要表达在胞质,且在6 h达高峰。UVB辐照后,p-p53和p21蛋白表达不断上升,p21表达较滞后;D-Gal处理后,p-p53和p21表达分别在6 h和12 h达高峰;BLM刺激后,p-p53和p21蛋白表达呈同步上升趋势,BLM浓度越高,其达高峰时间越短。结论成功建立3种GC-1细胞DNA损伤模型,且D-Gal处理GC-1细胞DNA损伤较轻,而UVB辐照和BLM刺激DNA损伤较重。

Aim To establish three kinds of DNA damage models of mouse testicular spermatogonia cell line GC-1 cells and analyze their similarities and differences.Methods GC-1 cells were treated with UVB irradiation,D-galactose(D-Gal)or bleomycin(BLM),respectively.Then the expression and localization ofγ-H2AX were detected by Western blot and immunofluorescence,the expression and localization of 8-OHdG were measured by immunofluorescence,and the expression levels of p-p53 and p21 were measured by Western blot.Results The expression ofγ-H2AX in GC-1 cell reached to the peak 4 h after UVB irradiation and 6 h after D-Gal stimulation,whereasγ-H2AX expression gradually increased after BLM stimulation,and the higher the concentration of BLM,the shorter the time to reach the peak.The results of immunofluorescence showed that 8-OHdG expression was observed in the nucleus and cytoplasm of GC-1 cells after UVB irradiation and BLM stimulation,and the longer the culture time,the more the expression in the nucleus.In contrast,the expression of 8-OHdG was observed in the cytoplasm and reached the peak at 6 h in the D-Gal stimulated GC-1 cells.After UVB irradiation,the protein expression levels of p-p53 gradually increased,while p21 protein expression appeared later than that of p-p53;in the D-Gal stimulated GC-1 cells,the protein expression levels of p-p53 reached the peak at 6 h,and p21 protein expression reached the peak at 12 h;after low concentration BLM stimulation,the protein expression levels of p-p53 and p21continuously increased,and after high concentration BLM stimulation,the protein expression levels of p-p53 and p21 reached its peak at 2 h,then decreased at 4 h.Conclusions Three kinds of DNA damage models of GC-1 cells are successfully established,and the DNA damage in GC-1 cells treated with D-Gal is mild,while the DNA damage in GC-1 cells treated by UVB irradiation and BLM stimulation is more severe.