彗星试验检测DNA交联的研究

Detection of DNA crosslinks with comet assay

为了寻求一种简便、快速的DNA交联检测方法 ,本文以过氧化氢 (H2 O2 )为标准断裂剂 ,用彗星试验检测了甲醛 (formaldehyde,FA)诱导的TK6细胞的DNA交联作用 ,并对两种实验方案进行了比较。结果显示 :随着甲醛浓度的增加 ,由过氧化氢造成的DNA迁移距离减小 ;当甲醛浓度为 10 0 0 μmol L时 ,无一个细胞拖尾 ,提示甲醛完全抑制了过氧化氢诱导的DNA迁移。此外 ,两种实验方案均可检出甲醛的DNA交联作用 ,但以先加交联剂、后加断裂剂的方案优于先加断裂剂、后加交联剂的方案 ,因为前一方案操作更简便 ,不受DNA修复的影响 ,所测DNA交联的剂量—反应关系更好。因此 ,彗星试验确为一种灵敏、简便和快速的DNA交联检测方法。

A simple and rapid method for the detection of DNA cross links was explored. DNA cross links was induced by formaldehyde (FA) in TK6 cells. Hydrogen peroxide was used as a standard DNA breakage agent. Two protocols on detecting DNA cross links with comet assay were tested and compared. The results showed that with increasing FA, the length of DNA migration caused by H 2O 2 was decreased. When the level of FA was 1000 μmol/L, no any tailed cell could be observed, the migration of DNA was inhibited completely even H 2O 2 was used. The detection of DNA cross link could be tested by both protocols. However, when the DNA cross link agent (FA) was added to the cells before the treatment with DNA breakage agent (H 2O 2), the result of the scheme was much better than that in the opposite order. The former scheme was much easy to be manipulated, with no influence from DNA repair and had better dose response relationship in the detection of DNA cross links. The comet assay is indeed a sensitive, convenient and rapid method for the detection of DNA cross link.