彗星电泳法检测H_2O_2对人淋巴细胞DNA损伤及其激光扫描共聚焦显微境下的观察和三维重建

INVESTIGATION ON DNA DAMAGE OF HUMAN LYMPHOCYTES BY H_2O_2 WITH COMET ASSAY AND OBSERVATION OF THE COMET'S 3-D STRUCTURE BY CONFOCAL MICROSCOPE

目的 :应用彗星电泳法于荧光显微镜和激光扫描共聚焦显微镜下观察人淋巴细胞经H2O2 作用后的DNA损伤情况。 方法 :人淋巴细胞经不同浓度H2O2(0、10、20、40、80μmol/L)作用20min,或20μmol/LH2O2 作用不同时间(0、10、20、30min)后 ,采用彗星电泳法 ,荧光显微镜下观察、照相 ,对尾长、总彗星长度、尾距进行测量和分析。同时采用激光共聚焦显微镜(LSCM)进行观察和三维重建 ;比较了不同染料(PI、AO、EB、Hochest33258)的染色效果。 结果 :H2O2 作用浓度为0、10、20、40、80μmol/L时 ,尾距( x- ±s)分别为0.001±0.002、0.31±0.35、1.23±0.96、2.82±1.38、3.52±0.96(P<0.01) ;作用时间为0、10、20、30min时 ,尾距( x- ±s)分别为0.05±0.09、0.22±0.30、1.62±0.93、1.89±1.09(P<0.01)。尾长、总彗星长度也有显著性增加 ,经统计学检验 ,P<0.01。与荧光显微镜相比 ,LSCM下的"彗星"轮廓十分清晰 ;PI和EB染色效果较好。 结论 :H2O2 能剂量依赖性和时间依赖性地导致人外周血淋巴细胞DNA的明显断裂损伤。利用LSCM构建"彗星"的三维图像将更有利于DNA损伤的准确测量。鉴于染色效果的差异 。

Purpose: To investigate DNA damage of human lymphocytes after treated with H2O2. Methods: Comet assay was employed to assess DNA damage of human lymphocytes treated with either various concentrations of H2 O2 (0、10、20、40、80 μmol/L) for 20 minutes or 20 μmol/L H2O2 for different time (0、10、20、30 minutes). To quantify the DNA damage, three different comet parameters were evaluated: the tail length, comet length and tail moment. Laser scanning confocal microscope (LSCM) was also introduced to investigate the 3-D distribution of DNA within the comet. Four fluorescent dyes including propidium iodide (PI), ethidium bromide (EB), acridine orange (AO) and Hochest33258 were compared. Results: The tail moments (-±s) were 0.001±0.002、0.31±0.35、1.23±0.96、2.82±1.38、3.52±0.96(P<0.01), respectively, after treatment with various concentrations of H2O2(0、10、20、40、80 μmol/L); the tail moments(-±s) were 0.05±0.09、0.22±0.30、1.62±0.93、1.89±1.09 (P<0.01), respectively, after treatment for different time(0、10、20、30 minutes ). Tail length and comet length also exhibited significant increases (P<0.01) with the increased dose and extended time. The 3-D reconstruction made by LSCM gave a more distinct image of the comet compared with the fluorescence microscope. PI and EB were better dyes in terms of the definition of the image. Conclusion: H2O2 induces dose-dependent and time-dependent cytotoxic effect in terms of DNA damage on human lymphocytes. The LSCM provides substantial and measurable improvement in the definition of the“comet'. PI and EB are suitable for comet assay.