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目的:探讨线粒体靶向抗氧化剂Mitoquinone(Mito Q)对冻融人精子的保护作用。方法:选取60份健康生育男性精液标本,每份精液一式6份,不含Mito Q者设为对照组(G0),而G1、G2、G3、G4、G5实验组混合液中分别含有2 nmol/L、20 nmol/L、200 nmol/L、2μmol/L、20μmol/L Mito Q,37℃孵育1 h后检测活性氧(ROS)、丙二醛(MDA)和线粒体膜电位(MMP)变化。选取合适Mito Q浓度B1、B2组用于精子冷冻保存,B0组未添加Mito Q,B1、B2组在精子冷冻保护液中分别含有200 nmol/L和2μmol/L Mito Q,进行冷冻保存,检测冷冻复苏后的ROS水平、MDA含量和MMP改变。结果:新鲜精液添加Mito Q孵育后,G3组和G4组前向运动精子百分率[(30.8±10.2)%和(32.7±13.5)%]和总活动率[(70.6±9.0)%和(70.3±11.9)%]显著高于G0组[(17.6±5.0)%、(54.9±11.5)%](P<0.05);随着Mito Q浓度的增加,ROS水平呈下降趋势,G3、G4、G5组(分别为86.5±31.6、93.6±42.0、45.1±15.0)显著低于G0组(160.8±39.7)(P<0.05);MDA含量G3、G4组[分别为(0.9±0.5)、(0.9±0.5)μmol/mg]明显低于G0组[(1.9±1.1)μmol/mg](P<0.05),而G5组[(1.7±0.7)μmol/mg]不但没有降低,反而显著高于G3、G4组(P<0.05);与G0组MMP(1 701±251)相比,G5组(1 156±216)显著降低(P<0.05),而G1、G2、G3、G4组(分别为1 810±298、1 995±437、1 950±334、1 582±314)无明显变化。冷冻复苏后各组前向运动精子百分率和总活动率均较新鲜精液明显下降(P<0.01),B1组前向运动精子百分率[(3.2±2.3)%]较B0组[(0.8±0.6)%]明显改善(P<0.05);B1组精子总活动率[(43.0±9.5)%]较B0组[(26.5±11.4)%]明显改善(P<0.05);B1组ROS[(34.6±12.3)]和B2组ROS[(37.0±10.5)]均较B0组[(56.9±14.3)]显著下降(P<0.05),B1组MDA[(1.4±0.5)μmol/mg]和B2组MDA[(1.4±0.6)μmol/mg]均较B0组[(2.6±1.0)μmol/mg]显著下降(P<0.05),B1组MMP[(1 010.0±131.5)]和B2组MMP[(880.6±128.6)]均显著高于B0组[(721.1±24.8)](P<0.05)。结论:在精液冻存液中添加200 nmol/L的Mito Q能有效提高人精子质量,可作为精液冷冻保护添加剂用于精液的冷冻保存。
Objective: To investigate the protective effect of mitochondria-targeted antioxidant Mitoquinone (Mito Q) on frozen-thawed human sperm. Methods: Sixty healthy sperm samples were collected from 6 male sperm samples, each of which contained 6 sperm samples. The control group (G0) contained no Mito Q, while the control group (G0) contained 2 nmol The changes of reactive oxygen species (ROS), malondialdehyde (MDA) and mitochondrial membrane potential (MMP) were detected by flow cytometry at 20 nmol / L, 200 nmol / L, 2μmol / L and 20μmol / . The appropriate concentrations of Mito Q were selected for B1 and B2 for sperm cryopreservation, Mito Q was not added for B0 group, and 200 nmol / L and 2 μmol / L Mito Q were separately contained in sperm cryoprotectant solution for B1, B2, ROS levels after freezing and thawing, MDA content and MMP changes. Results: The percentages of forward motile sperm [(30.8 ± 10.2)% and (32.7 ± 13.5)%] and total activity [(70.6 ± 9.0)% and (70.3 ± 11.9%] was significantly higher than that in G0 group [(17.6 ± 5.0)% vs (54.9 ± 11.5)%] (P <0.05). The ROS level decreased with the increase of Mito Q concentration. (P <0.05). The levels of MDA in G3 and G4 groups were (0.9 ± 0.5) and (0.9 ± 0.5, respectively) ) (μmol / mg) were significantly lower than those in G0 group (1.9 ± 1.1 μmol / mg) (P <0.05), while those in G5 group were significantly lower than those of G3 and G4 (P <0.05). Compared with G0 group (1 701 ± 251), G5 group (1 156 ± 216) decreased significantly (P <0.05), while G1, G2, G3 and G4 groups ± 298, 1995 ± 437, 1 950 ± 334, 1 582 ± 314). Compared with fresh semen, the percentage of motile sperm motility and the total motility of each group were significantly decreased after cryopreservation (P <0.01). The percentage of motile spermatozoa in group B1 [(3.2 ± 2.3)%] was significantly higher than that in group B0 [(0.8 ± 0.6) % (P <0.05). The total sperm motility in group B1 was significantly higher than that in group B0 [(43.0 ± 9.5)% vs (26.5 ± 11.4% (1.4 ± 0.5) μmol / mg] in group B1 and MDA in group B2 were significantly lower than those in group B0 [(12.3) and B2 [37.0 ± 10.5] (1.4 ± 0.6) μmol / mg] significantly decreased compared with B0 group (2.6 ± 1.0 μmol / mg) (P <0.05) ± 128.6)] were significantly higher than those in group B0 [(721.1 ± 24.8)] (P <0.05). Conclusion: Adding 200 nmol / L Mito Q to seminal fluid can improve the quality of human sperm and can be used as cryopreservation additive for cryopreservation of semen.