羟基自由基引起的脱氧核糖核酸损伤研究

摘要

以鲱鱼精脱氧核糖核酸(Herring sperm DNA)为研究对象,利用紫外光(UV,200～275 nn,66.4 Lx)激发纳米TiO2发生光催化作用介导产生羟基自由基(Hydroxyl radical,·OH),探讨·OH引发DNA氧化损伤特性.采用凝胶电泳和高效液相色谱(HPLC)分析法跟踪DNA损伤历程;应用电子自旋共振(Electron spin resonance,ESR)及分光光度法跟踪损伤过程氧化物种及H2O2相对浓度的变化;运用生物标准样8-9羟脱氧鸟苷(8-Hydroxy-2'-deoxyguanosine,8-OHdG)为内标物,通过HPLC分析DNA损伤产物,研究DNA损伤机理.结果表明,较单纯UV辐照或暗光(Dark)催化条件,DNA浓度10 mg/L,TiO2浓度1.5g/L、pH 7～8,紫外光激发纳米TiO2介导产生·OH引发DNA损伤程度最大;DNA损伤为·OH氧化历程.并伴随有深度氧化过程;DNA结构中鸟嘌吟最易氧化损伤,8-OHdG为DNA氧化损伤中间产物及鸟嘌吟氧化损伤的特异产物.%Oxidative damage characteristic of Herring Sperm DNA by hydroxyl radical ( · OH) which was excited by TiO2 under UV irradiation (200-275 nm, 66.4 Lx) was studied. The process of DNA damage was detected by gel electrophoresis and high performance liquid chromatographic analysis.Electron spin resonance (ESR) and spectrophotometry were used to determine the oxygen species and the relative concentration of H2O2 , respectively. The mechanism of DNA damage was studied using 8-hydroxy-2'-deoxyguanosine (8-OHdG) as an internal standard. The results showed that DNA can be damaged by · OH generated from photocatalysis. Compared with UV or Dark/TiO2 system, the degree of DNA damage reached the maximum in the UV/TiO2 system under the condition of DNA concentration 10 mg/L, TiO2 concentration 1.5 g/L and pH 7-8. The damage was caused by the process of · OH oxidation of TiO2 under UV irradiation and deep mineralization of DNA took place.Guanine was easy to damage than the other groups in DNA. 8-OHdG was one intermediate product of DNA damage and a special product of guanine damage.