不同质量精子线粒体DNA拷贝数及完整性的比较研究

发布时间：2018-03-27 04:49

本文选题：线粒体DNA　切入点：男性不育　出处：《中国人民解放军医学院》2014年硕士论文

【摘要】：【背景】目前全世界有10%-15%的夫妇受到不孕不育的困扰，其中40%-50%是由于男方因素引起，，全球范围男子生殖能力呈明显下降趋势。导致男性不育的病因十分复杂，生育相关基因的缺陷是重要原因之一。线粒体是精子中唯一的细胞器，经典的理论认为线粒体通过氧化磷酸化产生三磷酸腺苷（adenosinetriphosphate，ATP）供给精子能量。虽然线粒体产能对精子运动和功能的重要性还存争议，但在异常精液中确实发现了精子线粒体DNA（mitochondrial DNA,mtDNA）的突变、缺失及精子结构的异常。近年来，关于精子mtDNA的研究引起人们的广泛关注，但研究主要集中在基因的突变与缺失上，拷贝数的研究较少，不过也有学者提出可以通过测定mtDNA的拷贝数评估线粒体的功能状态。【目的】本研究的目的是通过比较不同质量精子mtDNA拷贝数及完整性差异，探讨精子mtDNA拷贝数和完整性与精子质量的关系，以及精子mtDNA对男性生育能力的影响。【方法】实时荧光定量PCR(Real-time quantitative Polymerase Chain Reaction，Real-time PCR)技术是一项已经发展成熟的研究手段，广泛应用于分子生物学和细胞生物学研究领域，具有快速、灵敏、准确的特点，能定量检测目的基因拷贝数及表达量的变化。本实验采用Percoll非连续密度梯度离心法将精液样本分为30%层精子和80%层精子，应用Real-time PCR技术定量分析不同质量精子各层mtDNA拷贝数。同时应用长链PCR技术测定不同质量精子80％层mtDNA完整性。【结果】52例精液样本中，正常组30%层精子mtDNA拷贝数（4.85±3.00）与其80%层精子（0.80±0.45）比较，差异有统计学意义（P0.01）；弱精子症组30%层精子mtDNA拷贝数（1.92±1.29）与其80%层精子（0.70±0.52）比较，差异有统计学意义（P0.01）；少精子症组30%层精子mtDNA拷贝数（13.33±9.96）与其80%层精子（1.52±0.68）比较，差异有统计学意义（P0.01）；少弱精子症组30%层精子mtDNA拷贝数（5.34±4.62）与其80%层精子（1.93±0.65）比较，差异有统计学意义（P0.05）。在80%层精子中，少精子症组（1.52±0.68）与少弱精子症组（1.93±0.65）mtDNA拷贝数与正常组（0.80±0.45）比较，差异有统计学意义（P0.01）；少弱精子症组（9.33±2.71）mtDNA完整性与正常组（27.01±17.36）、弱精子症组（32.36±21.75）及少精子症组（17.63±7.64）比较，差异有统计学意义（P0.01）。在30%层精子中，少精子症组（13.33±9.96）和弱精子症组（1.92±1.29）mtDNA拷贝数与正常组（4.85±3.00）比较，差异有统计学意义（P0.05）。比较80%层精子各参数相关性， mtDNA拷贝数与mtDNA完整性呈负相关（r=-0.6261,P0.01），mtDNA拷贝数与精子密度呈负相关（r=-0.6345,P0.01），mtDNA完整性与精子密度呈正相关（r=0.5842,P0.01）。【结论】质量差的精子中存在mtDNA拷贝数增加及完整性下降，mtDNA拷贝数的检测可能成为评估精子质量的一个指标。
[Abstract]:[background] at present, 10 to 15 percent of couples in the world are suffering from infertility, of which 40 to 50 percent are caused by male factors, and there is a marked downward trend in men's reproductive ability worldwide. The causes of male infertility are very complex. One of the important reasons is the defect of fertility related genes. Mitochondria are the only organelles in spermatozoa. The classic theory is that mitochondria provide sperm energy through oxidative phosphorylation to produce adenosine triphosphate ATP, although the importance of mitochondrial productivity to sperm motility and function remains controversial. However, the mutations, deletions and structural abnormalities of sperm mitochondrial DNA(mitochondrial DNA have been found in abnormal semen. In recent years, the research on sperm mtDNA has attracted wide attention, but the studies mainly focus on gene mutations and deletions. There are few studies on copy number, but some researchers suggest that mitochondrial function can be evaluated by measuring copy number of mtDNA. [objective] to study the relationship between sperm mtDNA copy number and sperm quality and the effect of sperm mtDNA on male fertility by comparing the difference of mtDNA copy number and integrity of sperm with different quality. [methods] Real-time fluorescence quantitative PCR(Real-time quantitative Polymerase Chain reaction Real-time PCRs is a mature research method, which is widely used in molecular biology and cell biology, and has the characteristics of fast, sensitive and accurate. The sperm samples were divided into 30% layer sperm and 80% sperm layer by Percoll discontinuous density gradient centrifugation. Real-time PCR technique was used to quantitatively analyze the mtDNA copy number of different sperm layers, and long chain PCR technique was used to determine the mtDNA integrity of different sperm layers. [results] in 52 semen samples, the mtDNA copy number of 30% layer sperm in normal group (4.85 卤3.00) was significantly different from that in 80% layer sperm (0.80 卤0.45), and the copy number of mtDNA in 30% layer sperm in asthenospermia group (1.92 卤1.29) was higher than that in 80% layer sperm (0.70 卤0.52). The mtDNA copy number of 30% layer sperm in oligozoospermia group (13.33 卤9.96) was significantly higher than that in 80% layer sperm layer (1.52 卤0.68), and the mtDNA copy number of 30% layer sperm in oligozoospermia group (5.34 卤4.62) was significantly higher than that in oligozoospermia group (1.93 卤0.65). The difference was statistically significant (P 0.05). The copy number of 0.65)mtDNA in oligozoospermia group (1.52 卤0.68) and oligozoospermia group (1.93 卤0.45) was significantly higher than that in normal group (0.80 卤0.45). There was significant difference between oligozoospermia group (9.33 卤2.71)mtDNA) and normal group (27.01 卤17.36), oligozoospermia group (32.36 卤21.75) and oligozoospermia group (17.63 卤7.64). The copy number of 1.92 卤1.29)mtDNA in oligozoospermia group (13.33 卤9.96) and asthenospermia group (1.92 卤3.00) was compared with that in normal group (4.85 卤3.00). There was a negative correlation between the mtDNA copy number and the integrity of mtDNA. There was a negative correlation between the mtDNA copy number and the sperm density. There was a positive correlation between the mtDNA integrity and the sperm density. [conclusion] the detection of mtDNA copy number in spermatozoa with poor quality may be an index to evaluate sperm quality by increasing the copy number of mtDNA and decreasing the integrity of mtDNA.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.6

【参考文献】