發(fā)布時間：2018-07-06 09:18

  本文選題：Y染色體微缺失 + ESR2基因��； 參考：《鄭州大學》2014年碩士論文

【摘要】：研究背景和目的 由世界衛(wèi)生組織(WHO)規(guī)定,夫婦生活1年以上并且沒有采用任何避孕措施,仍然無法生育的稱為不孕不育,由男方因素造成的稱為不育癥。近年來,不孕不育的比例在已婚的適齡夫婦中越來越高,并且,不孕不育給社會帶來了很多負面影響。男性不育癥中,病因很多并且相當復雜,比如外傷、睪丸發(fā)育不良、內(nèi)分泌或免疫性疾病、梗塞、服用激素藥物或腫瘤藥物、環(huán)境毒物、不良生活習慣以及遺傳因素等等。還有少數(shù)男性不育不能給出基于分子水平的合理解釋,被稱為特發(fā)性男性不育,這其中約有50%是由遺傳因素造成的。近年來,大量研究發(fā)現(xiàn),染色體異常、Y染色體微缺失及相關(guān)基因的突變都與男性生殖能力的改變有關(guān)。 越來越多的學者驗證了Y染色體長臂遠端AZF區(qū)域微缺失與精子發(fā)生障礙有重要關(guān)系。基于這一結(jié)論,要想研究相關(guān)基因多態(tài)性與男性生殖能力的相關(guān)關(guān)系,必須先排除Y染色體AZF微缺失的影響。 1996年,Mosselman等人首次從睪丸組織中克隆出了雌激素受體的另一種亞型,雌激素受體β (Estrogen receptor β)后來被稱為ERβ即ESR2。研究發(fā)現(xiàn),ESR2基因rs4986938G/A位點位于ESR2基因的第8外顯子的3’非編碼區(qū)第1730號核苷酸處。而rs1256049G/A位點則位于ESR2基因的第5外顯子的配體結(jié)合區(qū)的第1082號核苷酸處。2010年,Mohammad Reza Safarinejad等人對伊朗人群ESR2基因這兩個位點多態(tài)性與男性不育的相關(guān)性研究發(fā)現(xiàn),單倍型與男性不育有關(guān)系。2011年臺灣學者Wen Lee等人的研究發(fā)現(xiàn),ESR2基因的rs4986938、rs1256049位點的多態(tài)性與精液濃度有關(guān),rs1256049位點的多態(tài)性還與精子運動能力有關(guān)。2012年日本學者Tsutomu Ogata等對ESR2基因的多個SNPs位點多態(tài)性與男性不育的相關(guān)性研究中顯示,單倍型“TGTAGA"可能是男性不育的風險單倍型,但其中rs1256049位點的等位基因G/A在病例組和對照組中的頻率分布沒有顯著差異。目前,ESR2基因多態(tài)性與男性不育的相關(guān)性在不同研究報道中的結(jié)果不盡相同,并且國內(nèi)沒有這方面研究的相關(guān)報道,故本研究選取ESR2基因的rs4986938、rs1256049位點進行研究,擬分析ESR2基因多態(tài)性與河南漢族人群男性不育是否相關(guān)。 本研究首先用多重聚合酶鏈式反應(Mutiplex Polymerase chain reaction Mutiplex PCR)對研究對象的病例組進行了Y染色體AZF區(qū)域微缺失的檢測,排除AZF微缺失之后,對病例組剩下的研究對象再利用聚合酶鏈式反應-限制性片段多態(tài)性(Polymerase chain reaction restriction-fragment length polymorphism, PCR-RFLP)技術(shù)對河南漢族人群ESR2基因的rs4986938位點和rs1256049位點多態(tài)性進行分析,擬研究這兩個位點多態(tài)性在河南漢族人群中的分布情況及其與男性生殖能力的相關(guān)性,從而為男性不育的臨床檢測與治療提供相關(guān)理論依據(jù)。 研究對象： 病例組 收集2012年11月到2013年3月來鄭州大學第一附屬醫(yī)院門診檢驗科就診的397例不育癥患者的精液標本,患者年齡22-42歲(平均年齡29.4±6.1歲)�；颊呔鶕�(jù)世界衛(wèi)生組織(WHO)標準進行常規(guī)精液分析,并且連續(xù)三次檢查確診為無精子癥、少精子癥(精子密度20×106/m1)�；颊呔懦称魇中g(shù)、睪丸炎、精索靜脈曲張、內(nèi)分泌疾病、肝腎類疾病、染色體異常、生殖器異常等情況。 對照組 隨機選取通過正常手段生育至少一個健康孩子的健康男性347例作為對照組,年齡22-42歲(平均年齡31.7±2.6歲)。 所有病例組和對照組的研究對象均來自河南地區(qū),飲食結(jié)構(gòu)大致相同,并且都知情同意。 研究方法： 抽取精液用經(jīng)典的酚-氯仿方法提取基因組DNA,PCR擴增目的片段,再經(jīng)過Rsa Ⅰ和Alu Ⅰ限制性內(nèi)切酶酶切,最后用2.5%的瓊脂糖凝膠電泳對擴增片段和酶切片段進行基因型檢測,紫外分析凝膠成像系統(tǒng)觀察記錄。 統(tǒng)計學處理： 所有得到的數(shù)據(jù)都采用SHEsis在線軟件和SPSS17.0統(tǒng)計軟件進行分析,檢驗水準為：a=0.05。病例組和對照組均使用SHEsis在線軟件進行Hardy-Weinberg(HWE)檢測,以估計所選資料的人群代表性。使用SPSS17.0計算OR值以及95%置信區(qū)間,進行基因突變對男性不育發(fā)生的風險性估計。用在線軟件SHEsis進行單倍型分析。 結(jié)果： 1.檢測出397例病例組中有50例Y染色體微缺失。 2.河南漢族人群中,ESR2基因的rs4986938以及rs1256049位點基因型頻率都符合Hardy-Weinberg平衡,表明其具有良好的人群代表性。 3.本研究中,rs4986938位點在患者組和對照組對比中,基因型和等位基因頻率差異具有統(tǒng)計學意義(P0.05)。 4.對rs4986938位點進行的密度分組分析中,GG基因型在重度少精和輕度少精組間差異顯著(P0.05)。 5.在本研究中,對于rs1256049位點兩組之間的對比顯示：基因型和等位基因頻率差異具有統(tǒng)計學意義(P0.05)。 6.對rs1256049位點進行的密度分組分析,GG基因型在重度少精和極重、無精組間差異顯著(P0.05)。 7.在河南漢族人群中,對rs4986938G/A和rs1256049G/A兩個位點在男性不育組與正常對照組間進行單倍型分析,發(fā)現(xiàn)A-G、G-A、G-G單倍型在兩組間的差異有統(tǒng)計學意義(P0.05)。 結(jié)論： 1.ESR2基因rs4986938位點的A等位基因與rs1256049位點的A等位基因可能均是河南漢族人群男性不育的遺傳危險因子。 2.ESR2基因rs4986938和rs1256049位點的A-G/G-A單倍型可能是河南漢族人群男性不育的風險單倍型,G-G單倍型是男性不育的保護單倍型。 3.ESR2基因多態(tài)性可能與河南漢族男性不育的發(fā)生相關(guān)。
[Abstract]:Background and purpose of research
The WHO (WHO) stipulates that couples who have lived for more than 1 years and have not adopted any contraceptive measures, are still infertile, called infertility, and are called infertility caused by the male factors. In recent years, the proportion of infertility is getting higher and higher among married couples, and infertility has brought a lot of negative effects to the society. In male infertility, there are many and quite complex causes, such as trauma, dysplasia of the testicles, endocrine or immune diseases, infarction, taking hormone drugs or tumor drugs, environmental poison, bad habits and hereditary factors. Sexual infertility, about 50% of which are caused by genetic factors. In recent years, a large number of studies have found that chromosomal abnormalities, Y chromosome microdeletion and mutations in related genes have been associated with changes in male reproductive capacity.
More and more scholars have verified that the microdeletion of the distal AZF region of the long arm of the Y chromosome has an important relationship with the disturbance of spermatogenesis. Based on this conclusion, to study the correlation between genetic polymorphism and male reproductive ability, the effect of AZF microdeletion on the Y chromosome must be eliminated first.
In 1996, Mosselman and others first cloned another subtype of estrogen receptor from the testicular tissue. The estrogen receptor beta (Estrogen receptor beta) was later known as ER beta, or ESR2. study found that the ESR2 gene rs4986938G/A loci was located at the 3 'non coding region 1730th nucleotides of the eighth exon of the ESR2 gene. The relationship between the two loci polymorphism of the ESR2 gene in Iran population and the relationship between the two loci polymorphism of the ESR2 gene in Iran population and the male infertility in the number 1082nd nucleotides of the ligand binding region of the fifth exon of the ESR2 gene found that the haplotype and male infertility were related to the study of the ESR2 gene of the ESR2 gene of the Taiwan scholar Wen Lee, which was found by the Taiwan scholar in.2011. Rs4986938, the polymorphism of the rs1256049 locus is related to the concentration of semen. The polymorphism of the rs1256049 locus is also related to the motility of the sperm. The correlation between the polymorphisms of the multiple SNPs loci and male infertility of the ESR2 gene, such as Tsutomu Ogata of the Japanese scholar,.2012 years, shows that the haplotype "TGTAGA" may be the risk haplotype of male infertility. There is no significant difference in the frequency distribution of the allele G/A of the rs1256049 locus in the case group and the control group. At present, the correlation between the ESR2 gene polymorphism and male infertility is not the same in the different research reports, and there is no related report in this field at home. Therefore, this study selects the rs4986938, rs1256049 of the ESR2 gene. We studied the association between ESR2 gene polymorphism and male infertility in Henan Han population.
In this study, the Mutiplex Polymerase chain reaction Mutiplex PCR was used to detect the microdeletion of the Y chromosome AZF region in the case group of the subjects. After eliminating the AZF microdeletion, the remaining subjects of the case group were reused by the polymerase chain reaction restriction fragment polymorphism (Polymerase chain). Reaction restriction-fragment length polymorphism, PCR-RFLP) technique was used to analyze the rs4986938 and rs1256049 loci polymorphism of the ESR2 gene in Henan Han population. The distribution of the two loci polymorphism in the Han population in Henan and the correlation with male reproductive ability were studied, which was the clinical study of male infertility. The relevant theoretical basis is provided for the detection and treatment.
Research object:
Case group
The semen specimens of 397 infertile patients, aged 22-42 years (average age 29.4 + 6.1 years), were collected from November 2012 to March 2013 at the First Affiliated Hospital of Zhengzhou University. The patients were performed routine semen analysis according to the WHO (WHO) standard and were diagnosed as azoospermia and oligozoospermia in three consecutive cases. The disease (sperm density is 20 x 106/m1). Patients are excluded from genital surgery, orchitis, varicocele, endocrine diseases, liver and kidney diseases, chromosomal abnormality, and genital abnormalities.
control group
A total of 347 healthy men with normal health at least one healthy child were randomly selected as the control group, aged 22-42 years (mean age 31.7 + 2.6 years).
All cases and controls were from Henan, with similar dietary patterns and informed consent.
Research methods:
The extract semen was extracted by classical phenol chloroform method to extract genomic DNA, PCR to amplify the target fragment, and then Rsa I and Alu I restriction endonuclease was cut. Finally, the amplified fragment and enzyme cut fragment were detected by 2.5% agarose gel electrophoresis, and the UV analysis gel imaging system was observed.
Statistical treatment:
All the data obtained were analyzed using SHEsis online software and SPSS17.0 statistical software. The test level was that the a=0.05. case group and the control group all used SHEsis online software for Hardy-Weinberg (HWE) detection to estimate the representative of the selected data. The OR value and the 95% confidence interval were calculated by SPSS17.0, and the gene mutation was carried out. Risk assessment of male infertility. Haplotype analysis was performed using online software SHEsis.
Result:
1. out of 397 cases, 50 cases of Y chromosome microdeletions were detected.
2. of the Han population in Henan, the genotype frequencies of the rs4986938 and rs1256049 loci of the ESR2 gene all conformed to the Hardy-Weinberg balance, indicating that they have a good representation of the population.
3. in this study, there was a statistically significant difference in genotype and allele frequencies between the rs4986938 locus and the control group (P0.05).
4. in the density grouping analysis of rs4986938 locus, GG genotype was significantly different between severe oligozoospermia group and mild oligozoospermia group (P0.05).
5. in this study, the comparison between the two loci of rs1256049 locus showed that there was a statistically significant difference in genotype and allele frequency (P0.05).
6. according to the density grouping analysis of rs1256049 locus, GG genotype was significantly different between severe oligozoospermia and heavy inseminal group (P0.05).
7. in Henan Han population, the two loci of rs4986938G/A and rs1256049G/A were analyzed by haplotype between male infertility group and normal control group. It was found that the difference of A-G, G-A and G-G haplotypes between the two groups was statistically significant (P0.05).
Conclusion:
The A alleles at the rs4986938 loci of the 1.ESR2 gene and the A alleles at the rs1256049 locus may be the genetic risk factors for male infertility in Henan Han population.
The A-G/G-A haplotypes of the 2.ESR2 gene rs4986938 and rs1256049 loci may be the risk haplotypes of male infertility in Henan Han population, and the haplotype of G-G is the protective haplotype of male infertility.
3.ESR2 gene polymorphism may be associated with male infertility in Henan Han population.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698.2

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