摘要：
目的 建立核酸内切酶改良型体外彗星实验方法,并应用该方法检测DNA氧化损伤.方法 分别应用苯并[a]芘[B(a)P,20 μmol/L]、甲基磺酸甲酯[MMS,25 μg/ml]、秋水仙素(COL,5 mg/L)和长春新碱(VCR,0.5 mg/L)处理人支气管上皮(16HBE)细胞.采用噻唑蓝(MTT)实验评估细胞生存率,常规彗星实验和内切酶改良型彗星实验分别检测DNA损伤和氧化损伤,流式细胞术检测细胞内活性氧改变.结果 MTT实验显示,B(a) P、MMS、COL、VCR染毒后可引起较高水平的细胞内活性氧升高,4个组的细胞生存率分别为:(59.69±2.60)％、(54.33±2.81)％、(53.11±4.00)％、(51.43±3.92)％.常规彗星实验及甲酰胺嘧啶-DNA-糖基化(formamidopyrimidine-DNA-glycosylase,FPG)酶彗星实验均检测到B(a)P、MMS、COL、VCR引发的DNA损伤.FPG酶彗星实验中,Olive尾矩改变最明显,缓冲液组的B(a) P、MMS、COL、VCR组Olive尾矩分别为22.99±17.33、31.65±18.86、19.86 ±9.56和17.02 ±9.39,FPG酶处理后Olive尾矩分别为34.50±17.29、43.80±10.06、33.10±12.38和28.60±10.53,较缓冲液组分别增加58.94％、38.48％、66.86％和68.21％(t值分别为3.91、3.89、6.66和3.87,P值均＜0.05).相关性分析显示,Olive尾矩与细胞内活性氧也有较好的相关性(r=0.77,P＜0.05).结论 FPG酶改良型彗星实验可以有效的检测遗传毒物所致的DNA氧化损伤改变.%Objective The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines.Methods DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays.Cytotoxicity were assessed by MTT method.The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B (a) P),methyl methanesulfonate (MMS),colchicine (COL) and vincristine (VCR) respectively,and the dose is 20 μmol/L,25 mg/ml,5 mg/L and 0.5 mg/L for 24 h,respectively.Oxidative damage was also detected by levels of reactive oxygen species in treated cells.Results Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer.Cell survival rate were (59.69 ± 2.60) ％,(54.33 ± 2.81) ％,(53.11 ± 4.00) ％,(51.43 ± 3.92) ％ in four groups,respectively.There was the direct DNA damage induced by test genotoxicants presented by tail length,Olive tail moment (TM) and tail DNA (％) in the comet assay.The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it,and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand.In the three parameters,the Olive TM was changed most obviously after genotoxicants treatment.In the contrast group,the Olive TM of B(a) P,MMS,COL,VCR in the contrast groups were 22.99 ± 17.33,31.65 ± 18.86,19.86 ±9.56 and 17.02 ±9.39,respectively,after dealing with the FPG,the Olive TM were 34.50 ± 17.29,43.80 ± 10.06,33.10 ± 12.38,28.60 ± 10.53,increased by 58.94％,38.48％,66.86％ and 68.21％,respectively (t value was 3.91,3.89,6.66 and 3.87,respectively,and all P ＜ 0.05),and the correlation between Olive TM and reactive oxygen species was better than other parameters (r =0.77,P ＜ 0.05).Conclusion This study indicates that FPG-comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure,and the application of comet assay will be expanded.The endonuclease modified comet assay will be used widely in the toxicology and molecular epidemiology study.
