本文選題：線粒體DNA　切入點(diǎn)：男性不育　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年碩士論文

【摘要】：【背景】目前全世界有10%-15%的夫婦受到不孕不育的困擾，其中40%-50%是由于男方因素引起，，全球范圍男子生殖能力呈明顯下降趨勢(shì)。導(dǎo)致男性不育的病因十分復(fù)雜，生育相關(guān)基因的缺陷是重要原因之一。線粒體是精子中唯一的細(xì)胞器，經(jīng)典的理論認(rèn)為線粒體通過(guò)氧化磷酸化產(chǎn)生三磷酸腺苷（adenosinetriphosphate，ATP）供給精子能量。雖然線粒體產(chǎn)能對(duì)精子運(yùn)動(dòng)和功能的重要性還存爭(zhēng)議，但在異常精液中確實(shí)發(fā)現(xiàn)了精子線粒體DNA（mitochondrial DNA,mtDNA）的突變、缺失及精子結(jié)構(gòu)的異常。近年來(lái)，關(guān)于精子mtDNA的研究引起人們的廣泛關(guān)注，但研究主要集中在基因的突變與缺失上，拷貝數(shù)的研究較少，不過(guò)也有學(xué)者提出可以通過(guò)測(cè)定mtDNA的拷貝數(shù)評(píng)估線粒體的功能狀態(tài)。 【目的】本研究的目的是通過(guò)比較不同質(zhì)量精子mtDNA拷貝數(shù)及完整性差異，探討精子mtDNA拷貝數(shù)和完整性與精子質(zhì)量的關(guān)系，以及精子mtDNA對(duì)男性生育能力的影響。 【方法】實(shí)時(shí)熒光定量PCR(Real-time quantitative Polymerase Chain Reaction，Real-time PCR)技術(shù)是一項(xiàng)已經(jīng)發(fā)展成熟的研究手段，廣泛應(yīng)用于分子生物學(xué)和細(xì)胞生物學(xué)研究領(lǐng)域，具有快速、靈敏、準(zhǔn)確的特點(diǎn)，能定量檢測(cè)目的基因拷貝數(shù)及表達(dá)量的變化。本實(shí)驗(yàn)采用Percoll非連續(xù)密度梯度離心法將精液樣本分為30%層精子和80%層精子，應(yīng)用Real-time PCR技術(shù)定量分析不同質(zhì)量精子各層mtDNA拷貝數(shù)。同時(shí)應(yīng)用長(zhǎng)鏈PCR技術(shù)測(cè)定不同質(zhì)量精子80％層mtDNA完整性。 【結(jié)果】52例精液樣本中，正常組30%層精子mtDNA拷貝數(shù)（4.85±3.00）與其80%層精子（0.80±0.45）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）；弱精子癥組30%層精子mtDNA拷貝數(shù)（1.92±1.29）與其80%層精子（0.70±0.52）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）；少精子癥組30%層精子mtDNA拷貝數(shù)（13.33±9.96）與其80%層精子（1.52±0.68）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）；少弱精子癥組30%層精子mtDNA拷貝數(shù)（5.34±4.62）與其80%層精子（1.93±0.65）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。在80%層精子中，少精子癥組（1.52±0.68）與少弱精子癥組（1.93±0.65）mtDNA拷貝數(shù)與正常組（0.80±0.45）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）；少弱精子癥組（9.33±2.71）mtDNA完整性與正常組（27.01±17.36）、弱精子癥組（32.36±21.75）及少精子癥組（17.63±7.64）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）。在30%層精子中，少精子癥組（13.33±9.96）和弱精子癥組（1.92±1.29）mtDNA拷貝數(shù)與正常組（4.85±3.00）比較，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。比較80%層精子各參數(shù)相關(guān)性， mtDNA拷貝數(shù)與mtDNA完整性呈負(fù)相關(guān)（r=-0.6261,P0.01），mtDNA拷貝數(shù)與精子密度呈負(fù)相關(guān)（r=-0.6345,P0.01），mtDNA完整性與精子密度呈正相關(guān)（r=0.5842,P0.01）。 【結(jié)論】質(zhì)量差的精子中存在mtDNA拷貝數(shù)增加及完整性下降，mtDNA拷貝數(shù)的檢測(cè)可能成為評(píng)估精子質(zhì)量的一個(gè)指標(biāo)。
[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 鹵3.00) was significantly different from that in 80% layer sperm (0.80 鹵0.45), and the copy number of mtDNA in 30% layer sperm in asthenospermia group (1.92 鹵1.29) was higher than that in 80% layer sperm (0.70 鹵0.52). The mtDNA copy number of 30% layer sperm in oligozoospermia group (13.33 鹵9.96) was significantly higher than that in 80% layer sperm layer (1.52 鹵0.68), and the mtDNA copy number of 30% layer sperm in oligozoospermia group (5.34 鹵4.62) was significantly higher than that in oligozoospermia group (1.93 鹵0.65). The difference was statistically significant (P 0.05). The copy number of 0.65)mtDNA in oligozoospermia group (1.52 鹵0.68) and oligozoospermia group (1.93 鹵0.45) was significantly higher than that in normal group (0.80 鹵0.45). There was significant difference between oligozoospermia group (9.33 鹵2.71)mtDNA) and normal group (27.01 鹵17.36), oligozoospermia group (32.36 鹵21.75) and oligozoospermia group (17.63 鹵7.64). The copy number of 1.92 鹵1.29)mtDNA in oligozoospermia group (13.33 鹵9.96) and asthenospermia group (1.92 鹵3.00) was compared with that in normal group (4.85 鹵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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R711.6

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