少弱精子症患者精子候选差异蛋白Cu-Zn SOD和GSS的验证及其表达研究
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摘要
目的根据串联质谱标签(TMT)技术筛选少弱精子症患者精子差异蛋白,选取Cu-Zn SOD和GSS蛋白进行验证,建立候选蛋白标志物,并探讨二者的表达及意义。方法选择2017年7月-2018年4月在新疆医科大学第一附属医院生殖医学中心就诊的男性,收集105例少精子症(O组)、150例弱精子症(A组)、50例少弱精子症(OA组)和106例正常(N组)者的精液,分离精子,对少弱精子症患者精子进行差异蛋白质组学筛查,并在生物信息学分析基础上,选取Cu-Zn SOD和GSS蛋白,用免疫荧光和免疫印迹方法检测其在O组、A组、OA组和N组的表达量。结果少弱精子症患者精子,Cu-Zn SOD为下调差异蛋白(为N组的0.77倍),GSS为下调差异蛋白(为N组的0.82倍)。免疫荧光和免疫印迹结果显示,O组、A组和OA组精子Cu-Zn SOD蛋白的表达明显低于N组,差异有统计学意义(P<0.05)。OA组精子GSS蛋白表达明显低于N组,差异有统计学意义(P<0.05)。结论 Cu-Zn SOD和GSS蛋白与OA组蛋白质组学筛选结果一致,可作为少弱精子症男性精子的候选标志物。Cu-Zn SOD和GSS含量降低可能在少弱精子症患者精子氧化应激损伤过程中发挥重要作用,同时Cu-Zn SOD含量降低与少精、弱精子症患者精子质量改变相关。
Objective Objective The differentially expressed sperm proteins of oligoasthenospermia patients were screened by TMT technology. The proteins of Cu-Zn SOD and GSS were selected to validate the results. The candidate protein markers were established and their expression and significance were discussed. Methods The male patients were enrolled from July 2017 to April 2018 at the Reproductive Medicine Center of the First Affiliated Hospital of Xinjiang Medical University. Sperms were collected from 105 cases of oligospermia(O group), 150 cases of asthenospermia(A group), 50 cases of oligoasthenospermia group(OA group) and 106 cases of normal group(N group). Sperms were separated and differentially proteomic screening was carried out for oligoasthenospermia patients. On the basis of bioinformatics analysis, the expression of Cu-Zn SOD and GSS proteins were selected and detected by immunofluorescence and Western blot. Results In oligoasthenospermia goup, Cu-Zn SOD was down-regulated(0.77 times as much as that in group N) and GSS was down-regulated(0.82 times as much as that in group N). The results of immunofluorescence and Western blotting showed that the expression of Cu-Zn SOD protein in sperm of group O, group A and group OA was significantly lower than that of group N(P<0.05). The expression of GSS protein in sperm of OA group was significantly lower than that of N group(P<0.05). Conclusion The results of proteomic screening of Cu-Zn SOD and GSS proteins were consistent with those of OA proteomics, and could be used as candidate biomarkers for oligoasthenospermia. Decreased levels of Cu-Zn SOD and GSS may play an important role in oxidative stress damage of sperm in oligoasthenospermia, and the decrease of Cu-Zn SOD may be related to the change of sperm quality in oligospermia and asthenospermia.
Objective Objective The differentially expressed sperm proteins of oligoasthenospermia patients were screened by TMT technology. The proteins of Cu-Zn SOD and GSS were selected to validate the results. The candidate protein markers were established and their expression and significance were discussed. Methods The male patients were enrolled from July 2017 to April 2018 at the Reproductive Medicine Center of the First Affiliated Hospital of Xinjiang Medical University. Sperms were collected from 105 cases of oligospermia(O group), 150 cases of asthenospermia(A group), 50 cases of oligoasthenospermia group(OA group) and 106 cases of normal group(N group). Sperms were separated and differentially proteomic screening was carried out for oligoasthenospermia patients. On the basis of bioinformatics analysis, the expression of Cu-Zn SOD and GSS proteins were selected and detected by immunofluorescence and Western blot. Results In oligoasthenospermia goup, Cu-Zn SOD was down-regulated(0.77 times as much as that in group N) and GSS was down-regulated(0.82 times as much as that in group N). The results of immunofluorescence and Western blotting showed that the expression of Cu-Zn SOD protein in sperm of group O, group A and group OA was significantly lower than that of group N(P<0.05). The expression of GSS protein in sperm of OA group was significantly lower than that of N group(P<0.05). Conclusion The results of proteomic screening of Cu-Zn SOD and GSS proteins were consistent with those of OA proteomics, and could be used as candidate biomarkers for oligoasthenospermia. Decreased levels of Cu-Zn SOD and GSS may play an important role in oxidative stress damage of sperm in oligoasthenospermia, and the decrease of Cu-Zn SOD may be related to the change of sperm quality in oligospermia and asthenospermia.
引文
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[13] 李惠,张建芳.人类精液中的抗氧化防御系统[J].中国计划生育学杂志,2016,24(12) :850-854.
[14] 白双勇,王剑松,赵庆华.男性不育患者体重指数、精浆中谷胱甘肽过氧化物酶、活性氧与精液质量的关系[J].中国医科大学学报,2015,43(11):1038-1040.
[15] ZHENG J,PIAO M J,KIM K,et al.Fucoxanthin enhances the level of reduced glutathione via the Nrf2-mediated pathway in human keratinocytes[J].Marine Drugs,2014,12(7):4214-4230.
[2] 世界卫生组织.人类精液检查与处理实验室手册[M].5版.北京:人民卫生出版社,2011:17-20.
[3] CAMARGO M,INTASQUI P,CAMILA B D,et al.MALDI-TOF fingerprinting of seminal plasma lipids in the study of human male infertility[J].Lipids,2014,49(9):943-956.
[4] 王晓岚,罗德梅,巩晓芸.计算机辅助精液分析在不孕症诊疗中的应用价值探讨[J].新疆医科大学学报,2010,33(2):201-202.
[5] 黄若辉,邹晓峰,杨军.中西医结合治疗男性少弱精子症的系统评价[J].江西中医药大学学报,2014,26(2):69-72.
[6] ROBERT J,MARK A,BRETT N,et al.Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress?[J].Asian J Androl,2015,17(4):633-639.
[7] MORIELLI T,O′FLAHERTY C.Oxidative stress promotes protein tyrosine nitration and S-glutathionylation impairing motility and capacitation in human spermatozoa[J].Free Rad Biol Med,2012,53(7):S137-S147.
[8] HAMADA A,SANDRO C,ASHOK A.Unexplained male infertility potential causes and management[J].Human Androl,2011,1(1):2-16.
[9] 梁继仁,朱鸿雁,李翠珍,等.大鼠新生期苯并芘暴露对睾丸抗氧化酶活性和精子生成量的影响[J].环境与职业医学,2012,29 (5):290-293.
[10] 王祖龙,蒋平,孙自学,等.加味五子衍宗丸对实验性精索静脉曲张大鼠睾丸组织SOD,MDA的影响[J].中国实验方剂学杂志,2010,16(10):173-174.
[11] ZEINALI F,HOMAEI A,KAMRANI E.Sources of marine superoxide dismutases:Characteristics and applications[J].Int J Biol Macromol,2015,79:627-637.
[12] 张春香,秦小伟,郭丽娜,等.纳米锌水平对公羊精液品质、抗氧化酶活性及附睾Cu-ZnSOD表达的影响[J].中国农业科学,2015,48(1):154-164.
[13] 李惠,张建芳.人类精液中的抗氧化防御系统[J].中国计划生育学杂志,2016,24(12) :850-854.
[14] 白双勇,王剑松,赵庆华.男性不育患者体重指数、精浆中谷胱甘肽过氧化物酶、活性氧与精液质量的关系[J].中国医科大学学报,2015,43(11):1038-1040.
[15] ZHENG J,PIAO M J,KIM K,et al.Fucoxanthin enhances the level of reduced glutathione via the Nrf2-mediated pathway in human keratinocytes[J].Marine Drugs,2014,12(7):4214-4230.
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