Thr307Ala、Asn680Ser基因多态性与卵巢刺激反应差异的相关性及机制研究
发布时间：2018-10-10 20:43
【摘要】：研究目的：研究FSHR基因单核苷酸多态性Thr307Ala和Asn680Ser在中国汉族不孕症女性人群中的分布特征,探讨该遗传多态性对FSH刺激后卵巢反应的影响,并研究其与OHSS的关系；研究FSHR基因单核苷酸多态性Thr307Ala和Ser680Asn影响FSH刺激后卵巢反应的具体分子机制,并探讨这些多态是否影响FSHR的表达和功能。 研究方法：采用聚合酶链式反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)对450名不孕症女性患者进行FSHR基因Thr307Ala和Asn680Ser位点的基因型分析,检测月经第三天基础FSH水平、基础雌激素水平和基础LH水平；检测醋酸曲普瑞林控释注射剂降调节后的FSH水平、雌激素水平和LH水平；检测绒毛膜促性腺激素给药日的雌激素水平、LH水平和孕酮水平,阴道B超检查月经第三天基础窦卵泡数量、绒毛膜促性腺激素给药日卵泡数和排卵前卵泡数,B超引导经阴道穿刺取卵并记录获卵数。所得实验数据应用PHASE软件进行单倍型分析；应用Haploview软件进行连锁不平衡分析；应用方差分析、卡方检验和多元logistic回归分析进行统计学分析。体外使用定点诱变的方法构建不同基因型FSHR真核表达载体,并以HEK293细胞为模型建立野生型和不同突变型FSHR稳定表达细胞株。然后,分别利用半定量Real-Time PCR和Western Blotting技术检测上述多态是否影响FSHR的表达。最后,用ELISA检测上述突变对FSHR所介导cAMP水平的影响。研究结果： 1. FSHR基因Thr307Ala位点突变型AA(GG基因型)组的基础FSH(mIU/ml)水平[7.38±2.07vs6.34±1.75,6.63±1.94, P0.05,95%CI(6.75,8.01)]显著高于野生型TT (AA基因型)和突变杂合子型TA (AG基因型)组,且AA组的刺激天数显著高于TT和TA组；Ser680Asn位点突变型SS (GG基因型)组的基础FSH (mIU/mL)水平[7.51±2.01vs6.31±1.75,6.66±1.96, P0.05,95%CI (6.88,8.15)]显著高于野生型NN(AA基因型)和突变杂合子型NS(AG基因型)组,且SS组的刺激天数显著高于NN和NS组。 2.使用FSH后,携带FSHR基因Thr307Ala位点AA组和Ser680Asn位点SS组的不孕症女性患者发生卵巢低反应的比例均显著高于其他组,然而307和680位点基因型与OHSS的发生无统计学意义。 3. FSHR基因Thr307Ala和Ser680Asn多态性位点连锁不平衡分析显示D'=0.95,r2=0.84,有统计学意义。 4. FSHR Thr307Ala和Ser680Asn基因多态性在HEK293细胞系中不影响FSHR mRNA和蛋白表达水平,并且不同基因多态性在HEK293细胞系中对cAMP的分泌没有影响。 研究结论：FSHR基因Thr307Ala和Ser680Asn位点存在着近似完全的连锁不平衡。FSHR基因Thr307Ala和Ser680Asn位点影响不孕症患者的卵巢反应,与卵巢过度刺激综合征无显著相关。FSHR基因Thr307Ala和Ser680Asn多态性不影响FSHR蛋白的表达,与FSHR cAMP依赖途径无关。全文共计图12幅,表5个,参考文献68篇。
[Abstract]:Objective: to study the distribution of FSHR gene single nucleotide polymorphisms (Thr307Ala) and Asn680Ser in infertile women of Han nationality in China, to investigate the effect of the polymorphism on ovarian response after FSH stimulation, and to study the relationship between SNP and OHSS. To study the molecular mechanism of FSHR gene single nucleotide polymorphism (Thr307Ala) and Ser680Asn affecting ovarian response after FSH stimulation, and to investigate whether these polymorphisms affect the expression and function of FSHR. Methods: polymerase chain reaction-restriction fragment length polymorphism (polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP) was used to analyze the Thr307Ala and Asn680Ser loci of FSHR gene in 450 infertile women. Basal estrogen level and basal LH level; FSH level, estrogen level and LH level after controlled release injection of triplatin acetate were measured; estrogen level, LH level and progesterone level were detected on the day of administration of chorionic gonadotropin. The number of basic antral follicles on the third day of menstruation, the number of follicles on the day of administration of chorionic gonadotropin and the number of follicles before ovulation were examined by B ultrasound. The experimental data were analyzed by haplotype analysis using PHASE software, linkage disequilibrium analysis by Haploview software, statistical analysis by variance analysis, chi-square test and multivariate logistic regression analysis. The eukaryotic expression vectors of different genotypes of FSHR were constructed by site-directed mutagenesis in vitro, and the wild-type and different mutant FSHR expression cell lines were established using HEK293 cells as the model. Then, semi-quantitative Real-Time PCR and Western Blotting techniques were used to detect whether the polymorphism affected the expression of FSHR. Finally, ELISA was used to detect the effect of the mutation on the cAMP level mediated by FSHR. Results: 1. The basal FSH (mIU/ml) level in the FSHR gene Thr307Ala locus mutation AA (GG genotype group [7.38 卤2.07vs6.34 卤1.75 卤6.63 卤1.94, P0.05T95CI (6.75H8. 01)] was significantly higher than that in the wild-type TT (AA genotype and the mutant heterozygous TA (AG genotype, and the stimulation days in the AA group were significantly higher than those in the TT and TA groups. The level of basic FSH (mIU/mL) in the Ser680Asn locus mutant SS (GG genotype group [7.51 卤2.01vs6.31 卤1.75 卤6.66 卤1.96, P 0.05 ~ 95CI (6.88 ~ 8.15)] was significantly higher than that in the wild-type NN (AA genotype and the mutant heterozygous NS (AG genotype, and the stimulation days in SS group were significantly higher than those in NN and NS groups. After the use of FSH, the incidence of ovarian hyporesponse in infertile women with FSHR Thr307Ala locus AA group and Ser680Asn locus SS group was significantly higher than that in other groups. However, there was no significant difference in the occurrence of OHSS between 307 and 680 locus genotypes. The linkage disequilibrium analysis of Thr307Ala and Ser680Asn polymorphism loci of FSHR gene showed that there was significant difference between the two loci. FSHR Thr307Ala and Ser680Asn gene polymorphisms did not affect the expression of FSHR mRNA and protein in HEK293 cell line, and different gene polymorphisms had no effect on cAMP secretion in HEK293 cell line. Conclusion: the Thr307Ala and Ser680Asn loci of FSHR gene have nearly complete linkage disequilibrium. Thr307Ala and Ser680Asn loci of FSHR gene affect ovarian response in infertile patients. FSHR gene Thr307Ala and Ser680Asn polymorphism did not affect the expression of FSHR protein, but had no relationship with FSHR cAMP dependent pathway. The full text includes 12 figures, 5 tables and 68 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.6
本文编号：2263185
[Abstract]:Objective: to study the distribution of FSHR gene single nucleotide polymorphisms (Thr307Ala) and Asn680Ser in infertile women of Han nationality in China, to investigate the effect of the polymorphism on ovarian response after FSH stimulation, and to study the relationship between SNP and OHSS. To study the molecular mechanism of FSHR gene single nucleotide polymorphism (Thr307Ala) and Ser680Asn affecting ovarian response after FSH stimulation, and to investigate whether these polymorphisms affect the expression and function of FSHR. Methods: polymerase chain reaction-restriction fragment length polymorphism (polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP) was used to analyze the Thr307Ala and Asn680Ser loci of FSHR gene in 450 infertile women. Basal estrogen level and basal LH level; FSH level, estrogen level and LH level after controlled release injection of triplatin acetate were measured; estrogen level, LH level and progesterone level were detected on the day of administration of chorionic gonadotropin. The number of basic antral follicles on the third day of menstruation, the number of follicles on the day of administration of chorionic gonadotropin and the number of follicles before ovulation were examined by B ultrasound. The experimental data were analyzed by haplotype analysis using PHASE software, linkage disequilibrium analysis by Haploview software, statistical analysis by variance analysis, chi-square test and multivariate logistic regression analysis. The eukaryotic expression vectors of different genotypes of FSHR were constructed by site-directed mutagenesis in vitro, and the wild-type and different mutant FSHR expression cell lines were established using HEK293 cells as the model. Then, semi-quantitative Real-Time PCR and Western Blotting techniques were used to detect whether the polymorphism affected the expression of FSHR. Finally, ELISA was used to detect the effect of the mutation on the cAMP level mediated by FSHR. Results: 1. The basal FSH (mIU/ml) level in the FSHR gene Thr307Ala locus mutation AA (GG genotype group [7.38 卤2.07vs6.34 卤1.75 卤6.63 卤1.94, P0.05T95CI (6.75H8. 01)] was significantly higher than that in the wild-type TT (AA genotype and the mutant heterozygous TA (AG genotype, and the stimulation days in the AA group were significantly higher than those in the TT and TA groups. The level of basic FSH (mIU/mL) in the Ser680Asn locus mutant SS (GG genotype group [7.51 卤2.01vs6.31 卤1.75 卤6.66 卤1.96, P 0.05 ~ 95CI (6.88 ~ 8.15)] was significantly higher than that in the wild-type NN (AA genotype and the mutant heterozygous NS (AG genotype, and the stimulation days in SS group were significantly higher than those in NN and NS groups. After the use of FSH, the incidence of ovarian hyporesponse in infertile women with FSHR Thr307Ala locus AA group and Ser680Asn locus SS group was significantly higher than that in other groups. However, there was no significant difference in the occurrence of OHSS between 307 and 680 locus genotypes. The linkage disequilibrium analysis of Thr307Ala and Ser680Asn polymorphism loci of FSHR gene showed that there was significant difference between the two loci. FSHR Thr307Ala and Ser680Asn gene polymorphisms did not affect the expression of FSHR mRNA and protein in HEK293 cell line, and different gene polymorphisms had no effect on cAMP secretion in HEK293 cell line. Conclusion: the Thr307Ala and Ser680Asn loci of FSHR gene have nearly complete linkage disequilibrium. Thr307Ala and Ser680Asn loci of FSHR gene affect ovarian response in infertile patients. FSHR gene Thr307Ala and Ser680Asn polymorphism did not affect the expression of FSHR protein, but had no relationship with FSHR cAMP dependent pathway. The full text includes 12 figures, 5 tables and 68 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.6
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