DNA聚合酶β对细胞生物学特性及DNA损伤的影响

Effects of pol β on Biological Characteristics and DNA Damage in Mouse Embryonic Fibroblast

目的探讨DNA聚合酶β(polβ)的表达水平对细胞的生物学特性及重铬酸钾诱导的DNA损伤效应的影响。方法以polβ野生型(polβ+/+)、polβ缺陷型(polβ-/-)及polβ高表达型(polβoe)小鼠胚胎成纤维细胞为研究对象,采用四氮唑盐比色法绘制3种细胞的生长曲线;群体倍增实验观察3种细胞的倍增时间;HGPRT基因突变实验检测细胞的自发突变频率;同时用单细胞凝胶电泳实验分析3种细胞的自发DNA损伤和重铬酸钾诱导的DNA损伤效应的差异。结果 3种细胞生长曲线和群体倍增时间基本一致,细胞自发突变频率和自发DNA损伤差异也无统计学意义(P>0.05);随着重铬酸钾浓度的增加,3种细胞的拖尾率和尾长均增加,在相同重铬酸钾染毒浓度下polβ缺陷型细胞的DNA损伤明显大于polβ野生型和高表达型细胞,且以polβ高表达型细胞的DNA损伤效应最弱。结论 polβ表达水平对细胞的生长特征无明显影响,也不会造成细胞自发突变的增加;polβ基因缺陷使细胞对重铬酸钾诱导的DNA损伤更敏感,而高表达则对DNA损伤具有一定保护作用。

Objective To explore the effect of DNA polymerase beta（pol β） expression level on biological characteristics of mouse embryonic fibroblast（MEF） and the cellular response to DNA damage induced by potassium dichromate.Methods pol β wild-type cells（pol β ＋/＋）,pol β null cells（pol β-/-） and pol β overexpressed cells （pol β oe） were applied as a model system.The growth curve of cells was plotted by MTT assay; the doubling time of cells was detected by double time experiment;the spontaneous mutation frequency was determined by HGPRT gene mutation method and single cell gel electrophoresis assay（SCGE） was employed to observe the DNA damage either happened spontaneously or induced by potassium dichromate.Results Growth characteristic and doubling time of the three kinds of cells were similar and no obvious differences were found on spontaneous DNA damage and mutations frequency among them（P〉0.05）.Potassium dichromate increased comet rate and tail length in the three kinds of cells in a concentration dependent way.DNA damage of pol β-/-cells at the same dosage were more serious than the other cells both in comet rate and tail length（P〈0.05）.pol β oe cells demonstrated more resistant to DNA damage obviously than the others.Conclusion The expression level of pol β has no significant effect on the biological characteristic and spontaneous mutation frequency of MEF.pol β knock out cells is more sensitive to DNA damage induced by potassium dichromate,whereas,pol β over expression can help cells response to DNA damage and protect cells from death in a certain degree.