环磷酸鸟苷（cGMP）改善活性氧（ROS）对精子氧化应激损伤的初步研究

发布时间：2018-03-27 13:27

本文选题：环磷酸鸟苷　切入点：活性氧　出处：《兰州大学》2014年硕士论文

【摘要】：目的：(1)探讨活性氧(ROS)对精子活力、精子细胞死亡率、核DNA完整及顶体反应的影响；(2)探讨环磷酸鸟苷(cGMP)对ROS诱导的氧化应激对精子损伤的改善作用及其作用机理。方法：经Percoll梯度离心法制作精子悬液,应用计算机辅助分析系统(CASA)进行精子活力分析；应用伊红-Y染色法观察ROS对精子死亡率的影响；应用流式细胞仪(FACS)及ROS敏感的新型荧光探针H2DCF-DA检测精子细胞内活性氧的产生情况；应用RT-PCR检测精子细胞内抗氧化物酶的表达；应用AOT测定精子DNA的完整性；应用FITC-PSA荧光染色法检测精子顶体反应。结果：(1)100μM cGMP增加了精子的前向运动力；(2)200μM SNP对精子的前向运动力无影响,但与LY83583同时孵育精子时,可显著地降低精子的前向运动力；(3)过氧化氢酶(CAT)和cGMP可显著的逆转SNP和LY83583对精子前向性运动的抑制作用；(4)SNP和LY83583同时处理的精子死亡率明显上升,而且可生成大量的活性氧(ROS);(5) cGMP可以显著地诱导CAT和锰-超氧化物歧化酶(Mn-SOD)的表达,抑制了活性氧对精子细胞产生的氧化应激损伤；(6)精子的前向运动力随H202浓度的增加呈下降趋势,cGMP可以改善H202对精子前向运动力的抑制作用；(7)H202组DFI明显高于对照组,H2O2+cGMP组、H2O2+CAT组DFI均明显低于H202组,cGMP和CAT可以显著逆转H202对DNA完整性的损伤；(8)cGMP组顶体反应率明显高于对照组、H202组；H202组、SNP组的AR%均高于对照组。结论：(1)低浓度的NO、H2O2可以增加精子活力,高浓度时显著降低精子活力。(2)cGMP在保护精子免受氧化胁迫损伤中可能发挥着重要作用,能够降低ROS的产生、细胞死亡率和DNA完整性。(3)cGMP对抗氧化物酶的诱导表达可能参与了精子的氧化还原反应的调控。(4)cGMP可以促进精子顶体反应的发生。(5)cGMP对体外精子具有独特的生理作用,尤其对于ART的精子制备过程。
[Abstract]:Objective to investigate the effects of reactive oxygen species (Ros) on sperm motility, sperm cell mortality, nuclear DNA integrity and acrosome reaction. Methods: sperm suspension was prepared by Percoll gradient centrifugation, sperm motility was analyzed by computer aided analysis system (CASA), and the effect of ROS on sperm mortality was observed by eosin Y staining. The production of reactive oxygen species (Ros) in sperm cells was detected by flow cytometry (FACS) and a novel fluorescent probe (H2DCF-DA) sensitive to ROS. The expression of antioxidant enzymes in sperm cells was detected by RT-PCR. The integrity of DNA was determined by AOT. Sperm acrosome reaction was detected by FITC-PSA fluorescence staining. Results 100 渭 M cGMP increased the forward-motility of spermatozoa. 200 渭 M SNP had no effect on the forward motility of spermatozoa, but when incubated with LY83583, the sperm was incubated simultaneously. Catalase (catalase) and cGMP could significantly reverse the inhibitory effect of SNP and LY83583 on sperm anterior motility. The death rate of spermatozoa treated with SNP and LY83583 was significantly increased. Moreover, a large number of reactive oxygen species (Ros) 5) cGMP could significantly induce the expression of CAT and Mn-SOD. Inhibitory effect of Ros on oxidative stress injury induced by Ros) the forward motility of spermatozoa decreased with the increase of H202 concentration. CGMP could improve the inhibitory effect of Ros on sperm forward-motility. The DFI in the group of 7H202 was significantly higher than that in the control group. The DFI of H _ 2O _ 2 cGMP group was significantly lower than that of H _ 2O _ 2 CAT group and CAT group. The acrosome reaction rate in H _ 2O _ 2 cGMP group was significantly higher than that in H _ 2O _ 2 group and H _ 2O _ 2 group. Conclusion low concentration of H _ 2O _ 2 can increase sperm motility, and at high concentration, it may play an important role in protecting spermatozoa from oxidative stress, and can reduce the production of ROS, which may play an important role in protecting spermatozoa from oxidative stress. Cell death rate and the expression of antioxidant enzymes induced by DNA integrality may be involved in the regulation of redox reaction of spermatozoa. CGMP can promote the occurrence of acrosome reaction in spermatozoa. It has a unique physiological effect on spermatozoa in vitro. Especially for the sperm preparation process of ART.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.6

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