过氧化氢诱导的人正常和早老细胞中DNA损伤及其修复研究

The Study of Hydrogen Peroxide Induced DNA Damage and Recovery in Normal Aging and Premature Aging Human Cells

目的研究人早老细胞和正常衰老细胞在氧化应激条件下的DNA损伤和修复。方法采用免疫荧光技术和彗星电泳技术,分别检测3组不同群体倍增数(PD)的人正常二倍体成纤维细胞BJ(青年组第14代,成年组第30代,衰老组第45代)和2组不同PD的人赫-吉二氏综合征(HGPS)细胞(青年组第8代,衰老组第17代)的DNA基础损伤程度。研究过氧化氢诱导造成以上细胞组DNA损伤及去除致损因素正常培养后的修复水平,采用免疫荧光和彗星电泳技术检测细胞在沉默DNA损伤修复蛋白着色性干皮病蛋白A(XPA)表达前后的修复能力。结果 BJ细胞衰老组DNA损伤程度较高,与成年组相比,对DNA损伤诱导因子更加敏感(P<0.05);成年组与青年组相比,对损伤诱导因子也更敏感(P<0.05)。HGPS细胞青年组的DNA基础损伤程度即已达到衰老BJ细胞类似或更高水平,且与BJ细胞具有一致的DNA损伤年龄变化趋势。经siRNA沉默XPA表达后可部分恢复HGPS细胞的修复能力,对BJ细胞则没有影响。随年龄增长无论正常还是早老细胞,DNA损伤程度增加,修复效率降低。结论 XPA功能异常抑制了HGPS细胞的损伤修复。

Objective To study the DNA damage and recovery induced by hydrogen peroxide in normal aging and premature aging human cells.Methods The immunofluorescent assay and comet assay were used to estimate basal DNA damage in normal aging BJ cells and premature aging Hutchinson-Gilford progeria syndrome（HGPS） cells,which were divided into three and two distinct population doubling（PD） number groups（BJ 14,30,45 and HGPS 8,17） respectively.The DNA damage induced by hydrogen peroxide of these cell populations,as well as their repair activity,was also studied.Finally,the recovery capability before and after the xeroderma pigmentosum group A（XPA） knocked down in these groups was measured.Results Our results indicated that the normal BJ cells of older PD number showed higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than the adult one,who,in turn,was more sensitive than the younger ones.The basal damage level of HGPS cells was same or higher than the older BJ cells.HGPS cells also had the same consistent damage change as BJ cells after treatment.The decline of the repair efficiency with age to the DNA damage induced by the agent was also observed.Conclusion The DNA repair of HGPS is repressed by dysfunction of XPA.