乳酸脱氢酶C4活性检测及其与男性不育的关系

发布时间：2018-07-03 10:23

本文选题：乳酸脱氢酶C4 + 活性测定　；参考：《福建医科大学》2015年硕士论文

【摘要】：近几十年,全球范围内育龄男性的精液质量呈下降趋势,男性不育已成为世界性重要卫生健康问题。而男性不育中,除少部分病因已知外,60%~75%的原因并不明确,给不育症的临床诊疗带来极大困难。乳酸脱氢酶C4(lactate dehydrogenase C4,LDH-C4),是上世纪60年代由Goldberg等和另一个研究小组发现的乳酸脱氢酶同工酶之一,可催化丙酮酸和乳酸之间的可逆性反应。LDH-C4表达具有很高的组织特异性,主要存在于哺乳动物发育成熟的睾丸和精子中,是精子能量代谢的一个关键酶,与精子的生成、代谢及获能等过程有密切关系。本课题组曾提出:不明原因不育患者中,有一部分是由精子LDH-C4活性降低引起的,并且存在LDH-C4的表达下降,导致酶活性异常,从而影响精子的运动和功能,最终导致不育。目前国内缺少LDH-C4活性异常在男性不育诊断中的大样本比例统计。本研究检测了1000例不明原因不育男性的精子LDH-C4活性,计算活性异常患者的比例,发现LDH-C4活性降低患者约占所有不明原因不育患者的11.5%。同时,本研究初步探讨LDH-C4活性与患者其他有关临床指标的相关性,结果显示LDH-C4活性与精子活力呈正相关,而与精浆弹性硬蛋白酶、顶体酶活性、乙肝病毒DNA含量及精子ROS含量等无明显相关性。为探讨LDH-C4活性降低的原因,本研究采用实时定量PCR、免疫荧光以及Western blot技术,检测LDH-C4活性降低患者精子LDH-C4 m RNA及蛋白的表达水平,结果发现LDH-C4活性降低患者存在LDH-C4 m RNA和蛋白水平的表达下降。本研究采用草氨酸钠(sodium oxamate)特异性抑制LDH-C4活性,模拟精子LDH-C4活性异常状态,并通过计算机辅助精液分析系统(CASA)及精子获能试验,研究LDH-C4活性下降对精子运动及功能的影响。结果发现,LDH-C4活性缺失患者及活性抑制的精子均存在运动和获能异常。本研究进一步证明LDH-C4活性异常是导致男性不育的可能原因之一,为男性不育提供了新的病因学解释,为男性不育症的诊断和治疗增添了实验依据。
[Abstract]:In recent decades, the semen quality of men of childbearing age has declined worldwide, and male sterility has become an important health problem in the world. In male infertility, 75% of the causes are not clear, which brings great difficulties to the clinical diagnosis and treatment of infertility. Lactate dehydrogenase C4 (lactate dehydrogenase C4LH-C4, one of the lactate dehydrogenase isoenzymes discovered by Goldberg et al. And another research team in the 1960s, catalyzes the reversible reaction between pyruvate and lactic acid. The expression of LDH-C4 is highly tissue specific. It is a key enzyme of sperm energy metabolism, which is mainly found in the mature testis and sperm of mammals. It is closely related to the formation, metabolism and capacitation of spermatozoa. Our group has proposed that some of the patients with unexplained infertility are caused by the decreased activity of LDH-C4 in sperm, and there is a decrease in the expression of LDH-C4, which leads to abnormal enzyme activity, which affects the motility and function of spermatozoa, and finally leads to infertility. At present, there is a lack of large sample statistics of abnormal LDH-C4 activity in diagnosis of male infertility. In this study, the sperm LDH-C4 activity of 1000 unexplained infertile men was measured, and the proportion of patients with abnormal activity was calculated. It was found that the patients with decreased LDH-C4 activity accounted for about 11.5% of all unexplained infertility patients. The results showed that LDH-C4 activity was positively correlated with sperm motility, but positively correlated with seminal plasma elastase and acrosin activity. There was no significant correlation between DNA content of hepatitis B virus and Ros content of spermatozoa. In order to investigate the reasons for the decrease of LDH-C4 activity, the expression of LDH-C4 mRNA and protein in spermatozoa was detected by real-time quantitative PCR, immunofluorescence and Western blot. The results showed that the expression of LDH-C4 mRNA and protein was decreased in patients with decreased LDH-C4 activity. In this study, sodium oxalate (sodium oxamate) was used to specifically inhibit LDH-C4 activity and mimic the abnormal state of sperm LDH-C4 activity. The effects of decreased LDH-C4 activity on sperm motility and function were studied by means of computer-assisted semen analysis system (CASA) and sperm capacitation test. The results showed that there were abnormal motility and capacitation in patients with low LDH-C4 activity and spermatozoa with inhibition of LDH-C4 activity. This study further proves that the abnormal activity of LDH-C4 is one of the possible causes of male infertility, which provides a new etiological explanation for male infertility and provides experimental basis for the diagnosis and treatment of male infertility.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R698.2

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