检测DNA蛋白质交联的新方法.pdfVIP

HEREDITAS (Beijing) 2008 年5 月, 30(5): 659―662 ISSN 0253-9772 技术与方法 DOI: 10.3724/SP.J.1005.2008.00659 检测DNA-蛋白质交联的新方法 苏来, 宋宏宇 国家沈阳新药安全评价中心, 沈阳 110021 摘要: 将彗星实验进行改进以用于 DNA-蛋白质交联作用的检测。利用甲醛对受试动物肝细胞的影响来判定此 法是否适用于检测 DNA-蛋白质交联。由于在实验中添加一定量的蛋白酶 K, 可使单细胞在电泳时产生更大的 迁移, 因此可以利用添加蛋白酶K 前后的彗星尾距比来判断外来化合物对生物机体产生DNA 损伤效应的时候 是否有出现 DNA-蛋白质交联作用。结果表明, 该方法快速、经济、灵敏度较高, 可以在单细胞水平对甲醛等强 交联剂引发的不同组织的DNA-蛋白质交联效应进行检测, 希望该方法能成为指示DNA 交联能力的有用工具。 关键词: 甲醛; 肝细胞; DNA 断裂; DNA-蛋白质交联 Detection of DNA-protein crosslinks with modified comet assay SU Lai, SONG Hong-Yu National Research Center for New Drug Safety Evaluation, Shenyang 110021, China Abstract: Modifications of the comet assay were introduced to measure crosslinks and the effect of formaldehyde in liver cells of the tested animals was investigated by the new method to see whether the method is feasible. Since exposure of slides to proteinase K can increase DNA migration of the treated cells, the presence of DNA-protein crosslinks can be indi- cated by compare of the tail moment before and after the proteinase K added. The results showed that the modified protocol of the alkaline comet assay is a fast, inexpensive and sensitive tool for the detection of potent crosslinkers-induced DNA-protein crosslinks at single cell level. Due to its specific advantages, the modified comet assay seems to be a useful tool as a DNA crosslink potency indicator. Keywords: formaldehyde; liver cells; DNA breakage; DNA-protein crosslinks DNA 交联是外来化学物作用于 DNA 所致的重 用于检测 DNA 交联的报道[2~5], 而国内却很少见, 要遗传损伤, 与其他类型DNA 损伤相比, DNA 交联 几乎都是用 KCl-SDS 沉淀法进行检测, 但此方法费 较难修复或易于发生易错修复, 在细胞周期中维持 时且成本较高。本室在深人挖掘彗星实验的应用前 时间较长。DNA- 蛋白质交联(DPC) 的存在阻滞了 景时, 首次探讨了通过添加蛋白酶K 来检测