【作者】 李世珍；

【导师】 康山； 李琰；

【作者基本信息】 河北医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的:子宫内膜异位症(endometriosis,EMs,简称内异症)和子宫腺肌病(adenomyosis,简称腺肌病)是两种引起育龄期妇女盆腔疼痛和不孕的妇科常见病,是由基因和环境因素相互作用所致的遗传性疾病。人们普遍认为内异症由经血逆流引起,而腺肌病则由子宫内膜侵入子宫肌层所致。迄今为止,两病的发病机制均尚未完全明确。无论子宫内膜细胞异位到子宫体以外还是子宫肌层,异位的内膜细胞想要存活并发展为内异症或腺肌病,血管生成(angiogenesis)是其必要条件。而纤维细胞生长因子(fibroblast growth factors,FGFs)作为一种有强烈促增殖和分化作用的细胞生长因子,在血管生成过程中起着重要的作用。在FGF家族中,两个原型因子纤维细胞生长因子-1(Fibroblast growth factor-1,FGF1,acidic FGF,aFGF)和纤维细胞生长因子-2(Fibroblast growth factor-2,FGF2,basic FGF, bFGF)是最早被发现的,也是研究最多的两个细胞因子。存在于FGF1和FGF2基因上的单核苷酸多态性( single nucleotide polymorphisms,SNPs)可能通过影响其转录水平和蛋白质表达从而与一些疾病的发生发展相关。我们假设FGF1和FGF2基因上的SNPs位点可能通过改变其蛋白的表达水平进而影响内异症和腺肌病的发生发展。本研究通过病例-对照研究旨在探讨FGF1基因启动子区-1385A/G(C/T,rs:34011)和FGF2内含子1的754C/G(rs:2922979)单核苷酸多态性与中国北方汉族妇女子宫内膜异位症和子宫腺肌病发病风险的关系。方法:本研究采用病例-对照研究方法,以421例内异症患者、269例腺肌病患者和相应的健康对照个体为研究对象,收集静脉抗凝血各5ml,采用蛋白酶K消化-饱和氯化钠盐析法提取外周静脉血白细胞DNA,采用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法检测FGF1和FGF2基因多态位点的基因型频率分布。数据统计分析采用SPSS13.0软件包(SPSS Company, Chicago, Illinois, USA)进行,P<0.05认为有统计学意义。病例组与对照组的年龄差异行t检验。用Hardy-Weinberg平衡分析来评估对照组基因型频率分布,P>0.05认为对照组可代表整体人群。比较病例组与对照组中各位点基因型频率的分布并进行卡方检验。两个多态位点间的联合分析采用行×列表χ2检验。以非条件Logistic回归法计算相对风险度的比值比(odds ratio, OR)及95%可信区间(confidence interval, CI)。结果:1内异症和腺肌病组的年龄与对照组相比,差异均无统计学意义(P﹥0.05)。健康对照组中FGF1和FGF2两个多态位点基因型频率分布均符合Hardy-weinberg平衡(P﹥0.05)。2 FGF1 -1385A/G多态的AA、AG、GG基因型频率在内异症和对照组分别为7.8%、36.3%、55.8%和10.9%、41.1%、48.0%,两组相比无显著差异(P>0.05);但内异症患者中A等位基因型频率(26.0%)明显低于对照组(31.5%)(P<0.05);与-1385G/G基因型相比,A/A+A/G基因型可明显降低内异症的发病风险(OR=0.730,95%CI=0.557~0.958)。3腺肌病组FGF1 -1385A、G等位基因频率(28.4%和71.6%)与对照组(33.1%和66.9%)相比无显著性差异(P>0.05);腺肌病组A/A、A/G、G/G三种基因型频率分别为9.7%、37.5%和52.8%,与对照组(13.4%、39.4%和47.2%)相比也无差异(P>0.05);与G/G基因型相比,A/G和A/A基因型可能与腺肌病的发病风险无关, OR值分别为0.852(95%CI=0.593~1.225)和0.646(95%CI=0.370~1.129)。4 FGF2 754C/G多态C、G等位基因频率在内异症和对照组分别为94.1%、5.9%和90.1%、9.9%,两组相比有统计学意义(P<0.05);内异症组C/C、C/G、G/G的基因型频率(88.8%,10.5%和0.7%)与对照组(81.9%,16.4%和1.7%)相比差异有统计学意义(P<0.05);与C/C基因型相比,CG+GG基因型能明显降低内异症的发病风险( OR=0.570, 95%CI=0.385~0.844)。5腺肌病组FGF2 754C、G等位基因频率分别为92.0%和8.0%,与对照组(87.7%和12.3%)相比差异有统计学意义(P<0.05);腺肌病组C/C、C/G、G/G三种基因型频率分别为85.9%、12.3%和1.9%,与对照组(78.1%、19.3%和2.6%)相比无统计学差异(P>0.05);与C/C基因型相比,C/G+G/G基因型基因型能明显降低腺肌病的发病风险(OR=0.586, 95%CI=0.374~0.917)。6 FGF1和FGF2两个多态基因型联合分析显示,-1385G/G﹡754C/C联合基因型在人群中最常见;内异症组各种联合基因型与对照组相比有统计学差异(P<0.05);与-1385G/G﹡754C/C相比,-1385AA﹡754CG,-1385AA﹡754GG,-1385GA﹡754GG和-1385GA﹡754CG四种基因型可明显降低内异症的发病风险,OR值分别为0.601(95%CI=0.441~0.821),0.610(95%CI=0.393~0.946),0.778(95%CI=0.610~0.993)和0.731(95%CI=0.586~0.912);尽管腺肌病组各种联合基因型与对照组相比无明显差异(P>0.05),但与-1385G/G﹡754C/C相比,-1385AA﹡754CG基因型能明显降低腺肌病的发病风险, OR值为0.606(95%CI=0.422~0.869)。其它联合基因型并不能明显改变内异症和腺肌病的发病风险。结论:1 FGF1基因启动子区-1385A/G单核苷酸多态与子宫内膜异位症的发病风险有关,即A等位基因型的携带者患内异症的风险明显降低。2未发现FGF1基因启动子区-1385A/G多态与子宫腺肌病的发病存在关联。3 FGF2内含子1中754C/G单核苷酸多态与子宫内膜异位症和子宫腺肌病的发病风险相关,即CG+GG基因型可能是内异症和腺肌病发病的保护因素之一。4 FGF1和FGF2两个多态位点联合分析发现,-1385G/G﹡754C/C联合基因型在人群中最常见;与-1385G/G﹡754C/C联合基因型相比,携带-1385A/A或-1385G/A且同时携带754G/G或754C/G的联合基因型能明显降低内异症的发病风险;其中,-1385A/A﹡754C/G基因型还能明显降低腺肌病的发病风险;而其它基因型均不能改变两种疾病的发病风险。更多还原

【Abstract】 Objective : Endometriosis (EMs) and adenomyosis are common gynecological diseases, which can bring chronic pelvic pain and infertility for women of reproductive age, are produced by multiple gene loci interacting with each other and with the environment. It is the widely accepted that endometriosis is caused by the blood of menstruate countercurrented, while adenomyosis is caused by the endomembrane intrude into the muscular of the uterus. Their aetiology and pathogenesis are not completely understood to date. However, the endometrial cells implant either outside the uterine cavity or within the myometrial wall of the uterus, one of the preconditions must be the angiogenesis if implantation of these cells intends to be successful, in which the fibroblast growth factors (FGFs) play important roles. FGFs are potent factors during the proliferation and differentiation of endothelial cells. As two prototype factors, FGF1 (acidic FGF,aFGF) and FGF2 (basic FGF, bFGF) are the most extensively studied members because of their basic type in the FGFs. The single nucleotide polymorphisms (SNPs) of FGF1 and FGF2 may modify the transcriptional activity and protein expression of FGF1 and FGF2, and further contribute to the development of some diseases. We suppose that SNPs of FGF1 and FGF2 may influence the risk of developing endometriosis and adenomyosis by modifying their protein expression. The aim of the present study was to investigate association of the two SNPs, FGF1 -1385A/G (C/T, rs:34011) in the promoter and FGF2 754C/G (rs:2922979) in the intron 1, with the risk of endometriosis and adenomyosis in North Chinese women by case–control studies.Methods:This case-contral study included 690 patients (421 with endometriosis and 269 with adenomyosis) and frequency-matched healthy control women. Five milliliter of venous blood from each subject was drawn in Vacutainer tubes containing EDTA. The genomic DNA was extracted using proteinase K digestion followed by a salting out procedure. Genotypes of the FGF1 and FGF2 genes were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.Statistical analysis was performed using SPSS13.0 software package. A probability level of 5% was considered significant. The age difference of cases and frequency-matched controls was analyzed by the t-test. Hardy-Weinberg analysis was performed during the controls to analysis whether they can represent the whole population with the probability level of 5%. Comparison of the genotype and allelotype distribution of FGF1 and FGF2 respectively in patients and healthy controls was performed by means of Chi-square test. The combination genotype of FGF1 and FGF2 were analysed by two-sided contingency tables using Chi-square test. The odds ratio (OR) and 95% confidence Interval (CI) were calculated using an unconditional logistic regression model.Results:1 Age among endometriosis, adenomyosis and control women had no significant difference (P>0.05). The genotype frequencies of FGF1 and FGF2 in healthy controls did not significantly deviate from that expected for a Hardy-Weinberg equilibrium (P>0.05).2 The distribution of the FGF1 -1385A/A, A/G and G/G genotypes between endometriosis patients (7.8%, 36.3% and 55.8%, respectively) and controls (10.9%, 41.1% and 48.0%, respectively) had no significant difference (P>0.05). But the frequency of the A allelotype in endometriosis patients (26.0%) was significantly lower than that in the controls (31.5%) (P<0.05). Contrast to the -1385G/G genotype, the A/A+A/G allelotype could significantly decrease the risk of developing endometriosis significantly (OR=0.730; 95%CI=0.557~0.958).3 No significant difference about the FGF1 -1385A and G allele distribution was shown between adenomyosis patients (28.4% and 71.6%) and controls (33.1% and 66.9%) (P>0.05). Genotype frequencies of the FGF1 -1385A/A, A/G and G/G in the adenomyosis group were 9.7%, 37.5%, 52.8%, and 13.4%, 39.4%, 47.2% in the controls, respectively. There was not statistical difference between the two groups either (P>0.05). The -1385A/G and A/A genotype could not modify the risk of developing adenomyosis compared to the G/G genotype, the odds ratio was 0.852 (95%CI=0.593~1.225) and 0.646 (95%CI=0.370~1.129).4 The frequencies of the FGF2 754C and G allele among endometriosis patients and healthy controls were 94.1%, 5.9% and 90.1%, 9.9%, respectively; Significant difference about the two alleles distribution was shown between endometriosis patients and controls (P<0.05). The frequencies of the 754C/C, C/G and G/G genotypes among endometriosis patients (88.8%, 10.5% and 0.7%, respectively) were significantly different from those in healthy controls (81.9%, 16.4% and 1.7%, respectively) (P<0.05). Contrast to the C/C genotype, the C/G+G/G genotype could significantly decrease the risk of developing endometriosis (OR=0.570; 95%CI=0.385~0.844).5 The frequencies of the FGF2 754C and G allele among adenomyosis patients were 92.0% and 8.0%, which was statistical difference with the healthy controls (87.7% and 12.3%) (P<0.05). There was no difference about FGF2 754C/C, C/G, G/G genotypes between adenomyosis patients (85.9%, 12.3% and 1.9%) and healthy controls (78.1%, 19.3% and 2.6%) (P>0.05). However, compared with the C/C genotype, the C/G+G/G genotype could significantly decrease the risk of developing adenomyosis(OR=0.586; 95%CI=0.374~0.917).6 The combined analysis of FGF1 -1385A/G and FGF2 754C/G genotypes showed that the -1385G/G﹡754C/C was the most frequent combined genotypes in the population. There was statistical difference about the combined genotypes between endometriosis patients and healthy controls (P<0.05). Compared with the -1385G/G﹡754C/C genotype, -1385AA﹡754CG,-1385AA﹡754GG,-1385GA﹡754GG and -1385GA﹡754CG could significantly decrease the risk of developing endometriosis, the odds ratio were 0.601 (95%CI=0.441~0.821), 0.610 (95%CI=0.393~0.946), 0.778 (95%CI=0.610~0.993) and 0.731 (95%CI=0.586~0.912), respectively. In spite of no difference was shown about the combined genotypes between adenomyosis patients and healthy controls (P>0.05), but compared with -1385G/G﹡754C/C genotype, the -1385AA﹡754CG could significantly decrease the risk of developing adenomyosis, the odds ratio was 0.606 (95%CI=0.422~0.869). The other combined genotypes could not modify the risk of developing endometriosis and adenomyosis.Conclusions:1 The FGF1 -1385A/G SNP in the promoter region might be related to the risk of endometriosis development. The carriers of the A allelotype could significantly decreased the risk of developing endometriosis.2 No association was shown between the FGF1 -1385A/G SNP and the risk of developing adenomyosis.3 The FGF2 754C/G SNP in the intron 1 might be related to the risk of both endometriosis and adenomyosis development. The C/G+GG genotype might be one of the potential protective factors to the development of endometriosis and adenomyosis development.4 The combined analysis of FGF1 and FGF2 genotypes indicated that the -1385G/G﹡754C/C was the most frequent combined genotypes in the population. Compared with the -1385G/G﹡754C/C genotype, -1385AA﹡754CG, -1385AA﹡754GG, -1385GA﹡754GG and -1385GA﹡754CG could significantly decrease the risk of the endometriosis development. And the -1385A/A﹡754C/G combined genotype could significantly decrease the risk of developing adenomyosis too. The other genotypes could not modify the risk of developing endometriosis and adenomyosis.更多还原