彗星法分析紫外线诱导的人晶状体上皮细胞DNA的损伤及抗氧化剂对DNA损伤的拮抗作用

UV-induced DNA damage and protective effects of antioxidants on DNA damage in human lens epithelial cells studied with comet assay

目的探讨紫外线诱导的人晶状体上皮细胞（LEC）DNA损伤修复机制及抗氧化剂对DNA损伤的拮抗作用。方法采用照射剂量为0．0（对照组）及2．5、5．0、7．5、10．0mJ／cm^2（实验1～4组）的紫外线照射培养的人LEC，碱性彗星法（CA）分析人LEC DNA单链断裂（SSB）的程度和接受10．0mJ／cm^2紫外线照射后的自身修复情况，以及人LEC在10．0mJ／cm^2紫外线照射前后不同浓度抗氧化剂维生素C（VitC）、牛磺酸、超氧化物歧化酶（SOD）和表没食子儿茶素没食子酸酯（EGCG）对DNA损伤程度的影响。结果对照组与4个实验组DNA SSB程度呈上升趋势，5个组比较，差异有统计学意义（F=17．259，P〈0．01）；10．0mJ／cm^2紫外线诱导的人LEC的DNASSB后，其半修复时间为60min；紫外线照射前，加入抗氧化剂牛磺酸、SOD、EGCG，实验组和对照组比较差异有统计学意义（F值分别为6.591、13.542、4．626，P〈0．01），VitC对照组和实验组比较差异无统计学意义（F=1．451，P〉0．05）；紫外线照射后，加入抗氧化剂VitC、牛磺酸、SOD、EGCG，实验组和对照组比较差异有统计学意义（F值分别为6．571、4．810、6．824、9．182，P〈0．01）；紫外线照射前加入4种抗氧化剂，其组间效应的差异有统计学意义（F=8．870，P〈0．01），紫外线照射后加入4种抗氧化剂，其组间效应的差异无统计学意义（F=0．101，P〉0．05）。结论人LEC DNA的损伤程度与紫外线剂量呈线性关系；外源性VitC、牛磺酸、SOD、EGCG对紫外线照射诱导的人LEC DNA的损伤均有拈抗作用。紫外线照射前加入抗氧化剂的效应由强至弱依次为SOD、EGCG、牛磺酸、VitC，紫外线照射后加入4种抗氧化剂的效应差异无统计学意义。SOD和EGCG是高效抗氧化剂。

Objective To investigate the mechanism of UV-induced DNA damage and repair and the protective effects of antioxidants on DNA damage in human lens epithelial cells. Methods Human lens epithelial cells were irradiated at UV-doses 0.0 （ control group ）, 2.5, 5.0, 7.5, 10.0 mJ/cm^2（ treated group 1 -4）. The amounts of DNA single strand breaks （SSB） were measured with the alkaline comet assay （CA）. The spontaneous repair of DNA SSB after exposure to UV at 10.0 mJ/cm^2 was also determined in human lens epithelial cells. Human lens epithelial cells were treated with different concentration of VitaminC （ VitC ） , taurine, superoxide dismutase （ SOD ） and epigallocatechin gallate （ EGCG ） before and after ultraviolet radiation, the effects of antioxidants on DNA damage was examined with alkaline comet assay. Results The amount of DNA SSB in control group and treated groups 1 -4 showed increased tendency ,was dose-dependent to the dose of UV irradiation, the differences of DNA SSB in 5 group were significantly （ P 〈 0.01 ）. UV-induced DNA SSB at 10.0 mJ/cm^2 in human lens epithelial cells, the half repair time was 60 minutes. Human lens epithelial cells were treated with different concentrations of taurine, SOD and EGCG before ultraviolet radiation. The differences of DNA damage in control and various antioxidant treated groups was statistically significant （F = 6. 591,13. 542,4. 626 in cells treated with taurine, SOD and EGCG, respectively,P 〈 0.01 ）, the difference of VitC effect on DNA in control and treated group were not significantly （ F = 1.451, P 〉 O. 05 ）. Human lens epithelial cells were treated with different concentration of VitC, taurine, SOD and EGCG after ultraviolet radiation. The differences of DNA damage between the control and treated group were statistically significant （F =6.571,4. 810,6. 824,9. 182 in cells treated with VitC, taurine, SOD and EGCG, respectively, P 〈 0. 01 ）. The differences of protective effects on DNA damage in these four different kinds of antioxidants added before UV irradiation were statistically significant （P 〈 0. 01 ）. The differences of protective effects on DNA damage in these four different kinds of antioxidant added after UV irradiation were not significantly （ P 〉 0.05 ）. Conclusions UV irradiation has a dose- dependent effect on the DNA SSB of lens epithelial cells. Exogenesis VitC, taurine, SOD, EGCG possess protective effective to UV-induced DNA damage. SOD is one of the most powerful antioxidants if added before the UV irradiation and followed by EGCG,taurine and VitC orderly. Four kinds of antioxidants show no apparently differences added after UV-irradiation. SOD and EGCG both are powerful antioxidants.