SEMG1、SEPT12基因与弱精子症的相关性研究

发布时间：2018-03-24 13:05

本文选题：弱精子症SEMG1基因　切入点：SEPT12基因基因表达　出处：《郑州大学》2017年硕士论文

【摘要】：第一部分SEMG1、SEPT12基因的表达分析目的:检测精囊凝胶蛋白1(SEMG1)、胞裂蛋白12(SEPT12)基因在弱精子症(Asthenozoospermia,AZS)患者和正常对照精子中m RNA的表达,分析SEMG1、SEPT12基因与弱精子症的相关性。方法:收集成年男性正常对照组和弱精子症组精液样本各40例,采用Percoll密度梯度分离液纯化精子,提取精子RNA并将其逆转录为c DNA。采用实时定量RT-PCR(q RT-PCR)的方法,分别以对照组和弱精子症组精子的c DNA为模板,设计SEMG1、SEPT12基因的特异性扩增引物进行PCR扩增,比较两组精子中SEMG1、SEPT12基因m RNA表达差异。结果:通过qRT-PCR进行目的基因和内参基因的扩增,分别获得特异性扩增条带,SEMG1目的基因片段为163bp,SEPT12目的基因片段为167bp,内参基因GAPDH目的片段为189bp。对两样本组目的基因的相对表达量采用独立样本t检验分别进行统计分析。结果显示:1.AZS组SEMG1基因的m RNA表达水平(1.98±0.24)显著高于对照组(1.05±0.35),差异具有统计学意义(P0.05);2.AZS组SEPT12基因的m RNA表达水平(0.36±0.14)显著低于对照组(1.03±0.25),差异具有统计学意义(P0.05)。结论:1.精囊凝胶蛋白1基因(SEMG1)在弱精子症精子中表达上调,可能与AZS的发生有关;2.胞裂蛋白12基因(SEPT12)在弱精子症精子中表达显著降低,可能参与AZS的发生。第二部分SEMG1、SEPT12基因的多态性研究目的:应用KASP(Kompetitive Allele-Specific PCR)高通量SNP(Single nucleotide polymorphism)检测技术对SEMG1基因的rs16989820位点、rs2301366位点,及SEPT12基因的rs759991位点、rs6500634位点进行检测,初步探讨这四个位点的多态性与河南弱精子症患者的相关性,为弱精子症的诊断和治疗提供相关的理论依据。方法:收集276例正常成年男性精液样本作为对照组,276例弱精子症患者精液样本作为病例组,提取精子基因组DNA,通过KASP高通量SNP检测技术对SEMG1基因的rs16989820位点、rs2301366位点,及SEPT12基因的rs759991位点、rs6500634位点进行基因分型。随机选取各位点不同基因型个体的PCR扩增产物进行DNA测序验证。结果:1.SEMG1基因的rs16989820位点、rs2301366位点和SEPT12基因的rs759991位点、rs6500634位点在弱精子症组和对照组中的基因型分布频率符合Hardy-Weinberg平衡(P值均大于0.05),表明样本具有良好的人群代表性;2.SEMG1基因的rs16989820位点和rs2301366位点其等位基因和基因型频率在对照组和弱精子症组分布差异均无统计学意义(P0.05);3.SEPT12基因rs759991位点在弱精子症组和对照组间的等位基因和基因型频率分布差异显著,具有统计学意义(P0.05),携带T等位基因和TT基因型个体发生弱精子症的风险增高(P=0.007,OR=1.413,95%CI:1.098-1.818;P=0.021,OR=1.891,95%CI:1.097-3.261);4.SEPT12基因rs6500634位点在弱精子症组和对照组间T等位基因频率分布差异显著,具有统计学意义,携带T等位基因的个体发生弱精子症的风险降低(P=0.030,OR=0.661,95%CI:0.454-0.963);5.SEPT12基因rs759991和rs6500634位点的单倍型结果显示,携带C-T单倍型的个体可降低弱精子症的发生风险(P=0.006,OR=0.431,95%CI:0.233-0.798),而携带T-C单倍型的个体可增加弱精子症的发生风险(P=0.002,OR=1.549,95%CI:1.180-2.033)。结论:1.SEPT12基因rs759991位点T等位基因和TT基因型可能是河南汉族人群弱精子症的遗传危险因子,可增加弱精子症的发生风险;2.SEPT12基因rs6500634位点T等位基因可能是河南汉族人群弱精子症的遗传保护因子,可降低弱精子症的发生风险;3.SEPT12基因rs759991和rs6500634位点的C-T单倍型可能是弱精子症的保护单倍型,而T-C单倍型可能是弱精子症的风险单倍型。
[Abstract]:The first part of the SEMG1, SEPT12 gene expression analysis objective: detection of seminal vesicle gel protein 1 (SEMG1), cell splitting (SEPT12) protein 12 gene in asthenospermia (Asthenozoospermia, AZS) expression in patients and normal controls in M RNA sperm analysis SEMG1, SEPT12 gene and weak sperm correlation disease. Methods: to collect the adult male of normal control group and asthenospermia group semen samples in all 40 cases, purified sperm by Percoll density gradient separation, extraction of sperm RNA and its reverse transcription of C by real-time quantitative RT-PCR (DNA. Q RT-PCR) method, respectively in the control group and asthenospermia sperm group C DNA as template, design SEMG1, SEPT12 gene specific primer for PCR amplification, SEMG1 compared two groups of sperm, the differential expression of SEPT12 gene m RNA. Results: the target gene and reference gene were amplified by qRT-PCR, respectively by special amplified bands, SEMG1 The gene fragment of 163bp SEPT12 gene fragment of 167bp GAPDH gene fragment was 189bp. relative expression in two samples of target gene by independent samples t test were analysed. The results showed that m RNA 1.AZS group, the expression level of SEMG1 (1.98 + 0.24) was significantly higher than the control group (1.05 + 0.35), the difference was statistically significant (P0.05); m RNA 2.AZS group, the expression level of SEPT12 (0.36 + 0.14) was significantly lower than the control group (1.03 + 0.25), the difference was statistically significant (P0.05). Conclusion: 1. seminal vesicle gel protein 1 gene (SEMG1) expression in asthenospermia, may be related to with the development of AZS; 2. cell split protein 12 gene (SEPT12) significantly decreased expression in asthenospermia, may be involved in the pathogenesis of AZS. The second part of SEMG1, to study the polymorphism of SEPT12 gene: KASP (Kompetitive Allele-Specific PCR) Qualcomm The amount of SNP (Single nucleotide polymorphism) detection of SEMG1 gene rs16989820 locus, rs2301366 locus, rs759991 locus and SEPT12 gene, rs6500634 loci were detected to study the correlation between polymorphism and Henan asthenospermia at these four sites, provide theoretical basis for the diagnosis and treatment of asthenospermia. Collected 276 cases of normal adult male semen samples as control group, 276 cases of asthenospermia in patients with semen samples were selected as cases, extracting sperm genomic DNA by KASP high-throughput SNP detection technology rs16989820 locus of SEMG1 gene rs2301366 locus, rs759991 locus and SEPT12 gene, rs6500634 genotyping. Randomly selected members different genotypes of PCR were verified by DNA sequencing of PCR products. Results: the rs16989820 locus of 1.SEMG1 gene, rs2301366 gene and SEPT12 Rs759991 locus, rs6500634 genotype in asthenospermia group and control group in the frequency distribution with the Hardy-Weinberg equilibrium (P values were greater than 0.05), the results showed that the sample has a good representation of the population; the 2.SEMG1 gene rs16989820 locus and rs2301366 locus allele and genotype frequencies in different groups and asthenospermia group distribution there was no statistical significance (P0.05); 3.SEPT12 rs759991 gene significantly differences in asthenospermia group and control group between the genotype and allele frequency distribution was statistically significant (P0.05), the risk of carrying the T allele and TT genotype occurred in asthenospermia increased (P=0.007, OR=1.413,95%CI:1.098-1.818; P=0.021 OR=1.891,95%CI:1.097-3.261, 4.SEPT12); rs6500634 gene significantly differences in asthenospermia group and the control group T allele frequency distribution, with statistical significance, carrying T etc. The risk allele ontogeny of asthenospermia decreased (P=0.030, OR=0.661,95%CI:0.454-0.963); 5.SEPT12 gene rs759991 and rs6500634 loci haplotype showed that C-T haplotype individuals can reduce the risk of asthenospermia (P=0.006, OR=0.431,95%CI:, 0.233-0.798) and the T-C haplotype individuals can increase the risk of developing asthenospermia (P=0.002, OR=1.549,95%CI:1.180-2.033). Conclusion: 1.SEPT12 gene rs759991 T allele and TT genotype may be a genetic risk factor in Henan Han population asthenospermia, can increase the risk of asthenospermia; 2.SEPT12 gene rs6500634 T allele may be a genetic protective factor in Henan Han population asthenospermia, can reduce the occurrence the risk of asthenospermia; C-T haplotype of 3.SEPT12 gene rs759991 and rs6500634 loci may be weak sperm protection in single child Type, and T-C haplotype may be a risk haplotype of asthenospermia.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R698.2

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