Thr307Ala、Asn680Ser基因多態(tài)性與卵巢刺激反應(yīng)差異的相關(guān)性及機(jī)制研究
發(fā)布時間：2018-10-10 20:43
【摘要】：研究目的：研究FSHR基因單核苷酸多態(tài)性Thr307Ala和Asn680Ser在中國漢族不孕癥女性人群中的分布特征,探討該遺傳多態(tài)性對FSH刺激后卵巢反應(yīng)的影響,并研究其與OHSS的關(guān)系；研究FSHR基因單核苷酸多態(tài)性Thr307Ala和Ser680Asn影響FSH刺激后卵巢反應(yīng)的具體分子機(jī)制,并探討這些多態(tài)是否影響FSHR的表達(dá)和功能。 研究方法：采用聚合酶鏈?zhǔn)椒磻?yīng)-限制性片段長度多態(tài)性(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)對450名不孕癥女性患者進(jìn)行FSHR基因Thr307Ala和Asn680Ser位點的基因型分析,檢測月經(jīng)第三天基礎(chǔ)FSH水平、基礎(chǔ)雌激素水平和基礎(chǔ)LH水平；檢測醋酸曲普瑞林控釋注射劑降調(diào)節(jié)后的FSH水平、雌激素水平和LH水平；檢測絨毛膜促性腺激素給藥日的雌激素水平、LH水平和孕酮水平,陰道B超檢查月經(jīng)第三天基礎(chǔ)竇卵泡數(shù)量、絨毛膜促性腺激素給藥日卵泡數(shù)和排卵前卵泡數(shù),B超引導(dǎo)經(jīng)陰道穿刺取卵并記錄獲卵數(shù)。所得實驗數(shù)據(jù)應(yīng)用PHASE軟件進(jìn)行單倍型分析；應(yīng)用Haploview軟件進(jìn)行連鎖不平衡分析；應(yīng)用方差分析、卡方檢驗和多元logistic回歸分析進(jìn)行統(tǒng)計學(xué)分析。體外使用定點誘變的方法構(gòu)建不同基因型FSHR真核表達(dá)載體,并以HEK293細(xì)胞為模型建立野生型和不同突變型FSHR穩(wěn)定表達(dá)細(xì)胞株。然后,分別利用半定量Real-Time PCR和Western Blotting技術(shù)檢測上述多態(tài)是否影響FSHR的表達(dá)。最后,用ELISA檢測上述突變對FSHR所介導(dǎo)cAMP水平的影響。研究結(jié)果： 1. FSHR基因Thr307Ala位點突變型AA(GG基因型)組的基礎(chǔ)FSH(mIU/ml)水平[7.38±2.07vs6.34±1.75,6.63±1.94, P0.05,95%CI(6.75,8.01)]顯著高于野生型TT (AA基因型)和突變雜合子型TA (AG基因型)組,且AA組的刺激天數(shù)顯著高于TT和TA組；Ser680Asn位點突變型SS (GG基因型)組的基礎(chǔ)FSH (mIU/mL)水平[7.51±2.01vs6.31±1.75,6.66±1.96, P0.05,95%CI (6.88,8.15)]顯著高于野生型NN(AA基因型)和突變雜合子型NS(AG基因型)組,且SS組的刺激天數(shù)顯著高于NN和NS組。 2.使用FSH后,攜帶FSHR基因Thr307Ala位點AA組和Ser680Asn位點SS組的不孕癥女性患者發(fā)生卵巢低反應(yīng)的比例均顯著高于其他組,然而307和680位點基因型與OHSS的發(fā)生無統(tǒng)計學(xué)意義。 3. FSHR基因Thr307Ala和Ser680Asn多態(tài)性位點連鎖不平衡分析顯示D'=0.95,r2=0.84,有統(tǒng)計學(xué)意義。 4. FSHR Thr307Ala和Ser680Asn基因多態(tài)性在HEK293細(xì)胞系中不影響FSHR mRNA和蛋白表達(dá)水平,并且不同基因多態(tài)性在HEK293細(xì)胞系中對cAMP的分泌沒有影響。 研究結(jié)論：FSHR基因Thr307Ala和Ser680Asn位點存在著近似完全的連鎖不平衡。FSHR基因Thr307Ala和Ser680Asn位點影響不孕癥患者的卵巢反應(yīng),與卵巢過度刺激綜合征無顯著相關(guān)。FSHR基因Thr307Ala和Ser680Asn多態(tài)性不影響FSHR蛋白的表達(dá),與FSHR cAMP依賴途徑無關(guān)。全文共計圖12幅,表5個,參考文獻(xiàn)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 鹵2.07vs6.34 鹵1.75 鹵6.63 鹵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 鹵2.01vs6.31 鹵1.75 鹵6.66 鹵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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.6
本文編號：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 鹵2.07vs6.34 鹵1.75 鹵6.63 鹵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 鹵2.01vs6.31 鹵1.75 鹵6.66 鹵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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.6
【參考文獻(xiàn)】
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