抗氧化剂改善冻融人精子氧化应激性DNA损伤的研究  

Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure

作　　者：李志凌[1] 林琼林[1] 刘容菊[1] 谢文燕[1] 肖婉芬[1] 

机构地区：[1]汕头大学医学院第一附属医院生殖中心,515041

出　　处：《中华医学杂志》2007年第45期3174-3177,共4页National Medical Journal of China

基　　金：广东省科技厅基金(2005834201013)

摘　　要：目的探讨抗坏血酸盐、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)对冻融人精子氧化应激性 DNA 损伤的可能保护作用。方法 30份健康可生育男性精液分别添加改良人精子冷冻保护液,依所添加的抗氧化剂及终浓度分6组;检测各组冻融前后常规精子参数,精子核 DNA 完整性以及冻融后活性氧(ROS)水平。结果 (1)冻融后抗坏血酸盐300 μmol/L 组与 CAT 200 U、400 U 组a+b 级精子下降少于对照组(均 P<0.05),精子活率复苏率、ROS 水平则分别高于和低于对照组(67%±14%、68%±14%、69%±15%vs 59%±10%,均 P<0.05;30±13、30±11、30±11 vs 37±17,均 P<0.05)。(2)添加抗坏血酸盐300μmol/L 组,CAT 200 U、400 U 组的彗星细胞尾部 DNA 百分比及 Olire 尾矩与冷冻前相比,差异无统计学意义(P>0.05);但分别低于对照组(41%±4%、40%±7%、40%±6%vs 46%±6%,均 P<0.01;7.7±1.2、7.5±1.6、7.8±1.9 vs 10.1±3.1,均 P<0.01)。其余试验组上述指标则明显高于冷冻前(均 P<0.01),而与对照组差异无统计学意义(P>0.05)。(3)冻融后各实验组 a+b 级精子与 ROS 水平有负相关性(P<0.05或 P<0.01)。除抗坏血酸盐600 μmol/L 组外,其余试验组尾部 DNA%、Olive 尾矩则分别与其对应组 ROS 有显著正相关性(P<0.05、P<0.01)。结论在精液冷冻保护剂中添加一定浓度的抗坏血酸盐、CAT 可减少冷冻产生的过量 ROS,进而减轻后者对精子核 DNA 的氧化应激性损伤,从而提高冻融人精子质量。Objective To evaluate the protective effects of oxidative DNA damage by adding antioxidants： ascorbate, catalase （CAT）, and superoxide dismutase （SOD） in human semen samples undergoing cryopreservation procedure. Methods Semen sample form 30 fertile men were mixed with modified cryoprotectant and divided into six groups according to the category and concentration of antioxidants： ascorbate 300 μmoL/L, ascorbate 600 μmoL/L, CAT 200 U/ml, CAT 400 U/ml, SOD 200 U/ml, and SOD 400 U/ml. Comet assay was conducted to measure the percentage of comet cells, and the nuclear DNA damaged parameters： tail DNA percentage （TD%） and Olive tail moment （OTM）. Flow cytometry was used to detect the reactive oxidative species （ROS）. The motility （a ± b grade）, viable recovery rate, nuclear DNA integrity and reactive oxidative species （ROS） of all groups were analyzed before and/or after freeze-thawing. Results （1）After cryopreservation, compared with the control group, the a ± b grade sperm rates of the ascorbate 300 μmoL/L , CAT 200 U, and CAT 400 U groups were all higher than that of the control group （ all P 〈 0.05 ） , however, the levels of reactive oxygen species （ROS） of the ascorbate 300 μmoL/L , CAT 200 U, and CAT 400 U groups were 30 ±13,30 ± 11, and 30 ± 11 respectively, all significantly lower than that of the control group （ 37 ±17 , all P 〈 0.05 ）. The viable recovery rates of the ascorbate 300 μmoL/L , CAT 200 U, and CAT 400 U groups were 67% ±14% ,68% ±- 14% ,and 69% ±15% respectively, all significantly higher than that of the control group （59% ±10% , all P 〈 0.05）. （μ） The TD% levels of the ascorbate 300 μmoL/L, CAT 200 U, and CAT 400 U groups were 41% ±% ,40% ±% ,40% ±% , all similar to that of the raw semen（ all P 〉0.05）, but significantly lower than that of the control group（46% ±% , all P 〈0. 01 ）. The OTM levels of the ascorbate 300 μmoL/ L, CAT 200 U, and CAT 400 U groups were 7.7 ±1.2, 7.5 ±1.6 ,and 7.8 ±1.9, a

关 键 词：精液保存 冷冻 活性氧组分 过氧化氢酶 抗坏血酸盐 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]