Singh N P, Danner D B, Tice R R, Brant L, Schneider E L
Laboratory of Molecular Genetics, National Institute on Aging, Baltimore, MD 21224.
Mutat Res. 1990 May-Jul;237(3-4):123-30. doi: 10.1016/0921-8734(90)90018-m.
Previous biochemical studies on DNA repair competence and aging have been limited to techniques, such as alkaline elution or nucleoid sedimentation, involving mass cell populations. These techniques provide no information about the distribution of DNA damage and repair among individual cells and are unlikely to detect age-dependent changes affecting a minor fraction of the cell population. We have recently described a microgel electrophoretic assay (Singh et al., 1988) that measures, at the level of the individual cell, single-strand DNA breaks and alkali-sensitive sites. Here, we employ this method to analyze DNA damage and repair in lymphocytes isolated from the peripheral blood of 31 subjects (23 males and 8 females aged 25-91 years) and exposed in vitro to 200 rads of X-irradiation. While basal (pre-irradiation) levels of damage were independent of the age of the donor, an age-dependent increase in DNA damage was observed immediately following irradiation. For all subjects, the mean level of DNA damage was restored to pre-irradiation control levels within 2 h of incubation at 37 degrees C. However, a distribution analysis of DNA damage among cells within each sample indicated the presence of a few highly damaged cells (4-16%) in the 2-h sample, the occurrence of which was significantly more common among aged individuals. These data indicate an age-related decline in DNA repair competence among a small subpopulation of lymphocytes.
先前关于DNA修复能力与衰老的生化研究仅限于诸如碱性洗脱或核仁沉降等涉及大量细胞群体的技术。这些技术无法提供关于单个细胞中DNA损伤和修复分布的信息，并且不太可能检测到影响一小部分细胞群体的年龄依赖性变化。我们最近描述了一种微凝胶电泳测定法（辛格等人，1988年），该方法可在单个细胞水平上测量单链DNA断裂和碱敏感位点。在此，我们采用这种方法分析从31名受试者（23名男性和8名女性，年龄在25至91岁之间）外周血中分离出的淋巴细胞中的DNA损伤和修复情况，并将其在体外暴露于200拉德的X射线照射下。虽然基础（照射前）损伤水平与供体年龄无关，但在照射后立即观察到DNA损伤随年龄增长而增加。对于所有受试者，在37摄氏度孵育2小时内，DNA损伤的平均水平恢复到照射前的对照水平。然而，对每个样本中细胞间DNA损伤的分布分析表明，在2小时的样本中存在少数高度受损的细胞（4% - 16%），在老年个体中这种情况的发生明显更为常见。这些数据表明淋巴细胞的一个小亚群中DNA修复能力存在与年龄相关的下降。
