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目的研究大气可吸入颗粒物(PM10)致人肝细胞瘤细胞(HepG2)DNA损伤及其可能机制。方法单细胞凝胶电泳(SCGE)试验检测细胞DNA链断裂;2’,7’-二氢二氯荧光素(DCFH)法测定细胞内活性氧水平;免疫组化方法测定8-羟脱氧鸟苷(8-OHdG)在细胞内的表达水平;免疫细胞化学法检测细胞内核转录因子NF-κBp65蛋白表达水平。结果大连市4个监测点PM10有机提取物致HepG2细胞DNA链断裂作用存在地点和季节差异;7.5～30μg/mLPM10有机提取物作用HepG2细胞1h后,尾DNA%增大,呈剂量依赖关系。HepG2细胞与7.5～30μg/mL的PM10有机提取物接触1h后,可引起细胞内ROS水平表达的明显增加;7.5～30μg/mL的PM10有机提取物作用于HepG2细胞3h后,细胞内8-OHdG水平的表达增强;HepG2细胞与30μg/mL的PM10有机提取物接触24h后,NF-κBp65蛋白表达水平明显增高。结论PM10有机提取物可引起体外HepG2细胞DNA链断裂;DNA链断裂水平随不同季节和不同监测点而异;PM10引起DNA损伤的机制可能是细胞内ROS生成增多,8-OHdG表达增高及NF-κBp65蛋白表达水平升高而导致的氧化性DNA损伤。
Objective To study the DNA damage induced by inhalable particulate matter (PM10) in human hepatoma cell (HepG2) and its possible mechanism. Methods Single cell gel electrophoresis (SCGE) was used to detect the DNA strand breaks in cells. The intracellular reactive oxygen species (ROS) levels were measured by 2 ’, 7’-dichlorodithluorofluorescein (DCFH) and the levels of 8-hydroxydeoxyguanosine (8-OHdG) in the cells; immunocytochemistry was used to detect the expression level of NF-κBp65 protein. Results The DNA fragmentation of HepG2 cells induced by PM10 organic extracts from four monitoring sites in Dalian City was found to be different in location and season. The tail DNA% of HepG2 cells treated with 7.5-10 μg / mL PM10 organic extracts increased in a dose-dependent manner. HepG2 cells exposed to 7.5 ~ 30μg / mL PM10 organic extracts for 1h, the intracellular ROS levels can be significantly increased; PM10 organic extract of 7.5 ~ 30μg / mL in HepG2 cells 3h, 8-OHdG The expression of NF-κBp65 in HepG2 cells was significantly higher than that in HepG2 cells treated with 30μg / mL PM10 for 24 hours. Conclusion PM10 organic extract can cause DNA strand breaks in HepG2 cells in vitro. The DNA strand breaks vary with different seasons and different monitoring points. The mechanism of PM10-induced DNA damage may be the increase of intracellular ROS production, the increase of 8-OHdG expression and the increase of NF- Increased expression of κBp65 causes oxidative DNA damage.