【摘要】：男性不育是一種由遺傳，環(huán)境，飲食等諸多因素引起的多基因遺傳病。近年來(lái)隨著不孕不育癥發(fā)病率不斷升高，男性不育也逐漸成為全球最受關(guān)注的疾病之一。在人群中，大約有10%至15%育齡夫婦不能正常生育，其中大約有一半是男性不育所致。調(diào)查研究表明，男性人群中大約有4%至5%的個(gè)體患有遺傳因素所導(dǎo)致的男性不育。生精障礙是男性不育最為常見(jiàn)的病因之一，遺傳因素在生精障礙的發(fā)生過(guò)程中起著重要作用。近年來(lái)，在生精障礙遺傳病因的研究上取得了一定的進(jìn)展，發(fā)現(xiàn)了一些與生精障礙相關(guān)的遺傳因素，如染色體的畸形，Y染色體的微缺失以及某些基因的突變等。但這些因素只能解釋部分的病例，，仍有眾多的生精障礙的遺傳病因有待于進(jìn)一步闡明。精子發(fā)生是一個(gè)復(fù)雜的過(guò)程，包括精原細(xì)胞的增殖分化、減數(shù)分裂、精細(xì)胞分化成精子等幾個(gè)階段。這個(gè)過(guò)程中有眾多基因的參與，這些基因及其表達(dá)的異常都有可能導(dǎo)致生精障礙的發(fā)生。因此，這些參與生精過(guò)程的基因也被認(rèn)為是生精障礙重要的候選基因，研究這些候選基因與生精障礙的關(guān)系，可為男性不育遺傳病因、發(fā)病機(jī)制的最終闡明提供有價(jià)值的資料。 DNMT1(DNAmethyltransferase1)基因是近年來(lái)發(fā)現(xiàn)的一個(gè)生精障礙的重要候選基因，其編碼產(chǎn)物DNMT1是一種DNA甲基化酶，該酶在DNA的甲基化過(guò)程中發(fā)揮重要作用。研究表明，精子發(fā)生過(guò)程中涉及眾多基因的表達(dá)調(diào)控，而DNA甲基化是生精過(guò)程中基因表達(dá)調(diào)控的一種重要方式。某些基因的DNA甲基化是正常生精過(guò)程所必須的。在生精過(guò)程中，DNMT1基因在細(xì)胞增殖、減數(shù)分裂、精細(xì)胞分化等階段和成熟的精子中均有表達(dá)，主要負(fù)責(zé)催化和維持染色體復(fù)制后的DNA甲基化狀態(tài),提示其在正常的生精過(guò)程中可能有重要作用。在小鼠中研究顯示,DNMT1基因的異常表達(dá),會(huì)導(dǎo)致生殖細(xì)胞凋亡和生精障礙。在一些生精障礙患者中也發(fā)現(xiàn)了DNMT1基因的表達(dá)異常。因此,DNMT1基因可能與人類(lèi)生精障礙有關(guān),其基因變異可能在生精障礙中發(fā)揮作用。 近年來(lái),一些信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)與生精障礙的關(guān)系也逐漸受到關(guān)注。KIT/KITLG信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)被認(rèn)為是在生殖細(xì)胞正常發(fā)育調(diào)控中不可或缺的一個(gè)信號(hào)系統(tǒng)。KIT基因編碼產(chǎn)物KIT受體是一種酪氨酸激酶受體(tyrosine kinase receptor), KITLG(KIT ligand)基因的產(chǎn)物是KIT受體的特異配體KITLG。該信號(hào)系統(tǒng)通過(guò)KIT受體與KITLG的特異性結(jié)合而激活。在實(shí)驗(yàn)動(dòng)物中研究顯示,KIT/KITLG信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)的激活,對(duì)睪丸中生殖細(xì)胞的增殖、減數(shù)分裂、遷移、成熟等十分關(guān)鍵,在生精過(guò)程中發(fā)揮了重要作用。不同的動(dòng)物模型研究表明,KIT或KITLG基因的異常,會(huì)導(dǎo)致生精障礙和雄性不育。這些研究結(jié)果提示,KIT和KITLG基因的變異也可能與人類(lèi)生精障礙有關(guān)。 目前,尚未有DNMT1基因、KIT基因和KITLG基因的變異與人類(lèi)生精障礙的研究報(bào)道。有鑒于此,本研究采用聚合酶鏈反應(yīng)(polymerase chain reaction, PCR)、限制性片段長(zhǎng)度多態(tài)性(restriction fragment length polymorphism, RFLP)和DNA測(cè)序等技術(shù),在正常男性和無(wú)精癥及少精癥不育患者中,對(duì)DNMT1基因的SNP rs16999593,rs2228612, rs2228611, KIT基因的SNPrs3819392,以及KITLG基因的SNP rs995030, rs4474514的等位基因頻率和基因型頻率的分布進(jìn)行了研究和分析,初步了解這三個(gè)基因的變異與生精障礙的關(guān)系,為今后進(jìn)一步闡明這些基因在生精障礙發(fā)病過(guò)程中的機(jī)制提供一些參考依據(jù)。 具體研究結(jié)如下： 1.通過(guò)比較病例組與正常對(duì)照組間DNMT1基因SNP rs16999593, rs2228612,rs2228611的等位基因頻率和基因型頻率的分布,發(fā)現(xiàn)SNP rs16999593和rs2228611的等位基因和基因型頻率在少精癥患者和正常對(duì)照組間存在顯著性差異。SNP rs16999593的等位基因A(83.7%vs.77.6%, P=0.029, OR=1.47,95%CI=1.044-2.071)和基因型AA(69.1%vs.59%, P=0.027, OR=1.55,95%CI=1.045-2.314)以及SNP rs2228611的基因型AA(18.4%vs.9.9%,P=0.011,OR=2.05,95%CI=1.172-3.586)在少精癥患者中顯著高于正常對(duì)照組。這些結(jié)果表明,SNP rs16999593等位基因A和基因型AA,以及SNP rs2228611的基因型AA與少精癥的發(fā)病風(fēng)險(xiǎn)相關(guān),提示DNMT1基因的多態(tài)性可能影響少精癥的發(fā)病易感性。 2.對(duì)KIT基因的SNP rs3819392, KITLG基因的SNP rs995030和rs4474514的等位基因頻率和基因型頻率分布的研究結(jié)果顯示,KIT基因SNP rs3819392和KITLG基因SNP rs4474514這兩個(gè)位點(diǎn)的等位基因頻率或基因型頻率在少精患者和正常對(duì)照組間存在顯著性差異。KIT基因的SNP rs3819392的等位基因G(94.2%vs.90.0%, p=0.022)和基因型GG(89.2%vs.82.0%, p=0.042)在少精癥患者中顯著高于對(duì)照組；KITLG基因SNP rs4474514少精癥患者基因型CC頻率(8.2%vs.3.4%,p=0.034)也顯著高于對(duì)照組。這些結(jié)果表明KIT基因SNPrs3819392和KITLG基因SNP rs4474514的多態(tài)性可能與少精癥相關(guān)。
[Abstract]:Male infertility is a multi-gene genetic disease caused by many factors such as heredity, environment, diet and so on. In recent years, with the increasing incidence of infertility, male infertility has become one of the most common diseases in the world. In the population, about 10 to 15 per cent of the couples are not able to bear normal fertility, of which about half of them are male infertility. The survey shows that approximately 4% to 5% of the male population is male infertility due to genetic factors. Spermatogenesis is one of the most common causes of male infertility, and the genetic factors play an important role in the process of spermatogenesis. In recent years, some progress has been made in the study of the genetic disease of spermatogenic disorder, and some genetic factors related to the spermatogenic disorder, such as the abnormality of the chromosome, the microdeletion of the Y chromosome and the mutation of some genes, have been found. But these factors can only be explained in some cases, and there are still many genetic diseases of spermatogenic disorder that need to be further clarified. Spermatogenesis is a complex process, including the proliferation and differentiation of spermatogonia, meiosis, and the differentiation of sperm cells into sperm. There are a large number of genes involved in this process, and the abnormality of these genes and their expression can lead to the occurrence of spermatogenic disorders. Therefore, the genes involved in the spermatogenic process are also considered as important candidate genes of spermatogenic disorder, and the relationship between these candidate genes and the spermatogenic disorder can be studied, which can provide valuable data for the final elucidation of the pathogenesis of male infertility. DNMT1 (DNMT1) gene is an important candidate gene which has been found in recent years, its coding product DNMT1 is a kind of DNA methylase, which plays an important role in the process of DNA methylation. The study shows that the gene expression regulation is involved in the process of spermatogenesis, and the DNA methylation is an important part of gene expression regulation in the process of spermatogenesis. expression. DNA methylation of certain genes is necessary for normal spermatogenic processes In the process of spermatogenesis, the DNMT1 gene is expressed in the stages of cell proliferation, meiosis, and fine cell differentiation and the mature sperm, and is mainly responsible for catalyzing and maintaining the DNA methylation state after the chromosomal replication, indicating that the DNMT1 gene may play an important role in the normal spermatogenic process. In mice, the abnormal expression of DNMT1 gene can lead to germ cell apoptosis and spermatogenesis. The expression of DNMT1 gene was also found in some patients with spermatogenic disorder. Therefore, the DNMT1 gene may be related to the human spermatogenic disorder, and the genetic variation of the DNMT1 gene may play a role in the spermatogenic disorder. In recent years, the relationship between some signal transduction system and spermatogenic disorder is also gradually affected. The KIT/ KITLG signal transduction system is considered an integral part of the normal development and control of germ cells The KIT receptor is a tyrosine kinase receptor and the product of the KITLG (KIT ligand) gene is a specific ligand KI of the KIT receptor. TLG. The signal system is specifically combined with KITLG via KIT receptor The activation of KIT/ KITLG signal transduction system is critical to the proliferation, meiosis, migration and maturation of germ cells in the testis. The different animal model studies have shown that the abnormality of KIT or KITLG gene can lead to spermatogenic and male infertility. The results of these studies suggest that the variation of KIT and KITLG gene may also be related to human spermatogenesis. In the present, there are no DNMT1 gene, KIT gene and KITLG gene mutation and human spermatogenic disorder. In this study, the SNP rs16999593, rs2228612, rs228611, KIT gene of DNMT1 gene, rs2228612, rs228611 and KIT gene were used in the patients with normal male and azoospermia and oligospermia by using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and DNA sequencing. 19392 and the distribution of the allele frequency and genotype frequency of the SNP rs995030, rs4474514 of the KITLG gene and the distribution of the genotype frequency. The relationship between the variation of these three genes and the spermatogenic disorder was preliminarily studied, and the mechanism of these genes in the pathogenesis of spermatogenic disorder was further clarified. Some references. The results were as follows:1. The allele and genotype frequency of the SNP rs16999593 and rs228611 were found in the patients with oligospermia and the normal control by comparing the allele frequency and the genotype frequency of the DNMT1 gene SNP rs16999593, rs2228612 and rs228611 between the case group and the normal control group. There was a significant difference between groups. The allele A of the SNP rs16999593 (83.7% vs. 77.6%, P = 0.029, OR = 1.47,95% CI = 1.044-2.071) and genotype AA (69.1% vs.59%, P = 0.027, OR = 1.55,95% CI = 1.045-2.314), and the genotype AA of the SNP rs228611 (18.4% vs. 9.9%, P = 0.011, OR = 2.05,95% CI = 1.172-3.586) were among the patients with oligospermia The results showed that the SNP rs16999593 allele A and the genotype AA, and the genotype AA of the SNP rs228611 were related to the risk of oligospermia, suggesting that the polymorphism of the DNMT1 gene may affect 2. The study of the allele frequency and genotype frequency distribution of the SNP rs3819392, the KITLG gene, the SNP rs995030 and the rs4474514 of the KIT gene showed that the allele frequency or the genotype frequency of both the SNP rs3819392 and the KITLG gene SNP rs4474514 of the KIT gene were in less sperm and normal. There was a significant difference in the control group. The allele G (94.2% vs. 90.0%, p = 0.022) and genotype GG (89.2% vs. 82.0%, p = 0.042) of the KIT gene was significantly higher in the patients with oligospermia than in the control group; the genotype CC frequency of the KITLG gene SNP rs4474514 oligospermia (8.2% vs. 3.4%, p = 0.0 34) was also significantly higher in the control group. These results indicate that the KIT gene SNPrs3819392 and the KITLG gene SNP rs4474514
【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R698.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 凡時(shí)財(cái);張學(xué)工;;DNA甲基化的生物信息學(xué)研究進(jìn)展[J];生物化學(xué)與生物物理進(jìn)展;2009年02期

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