【作者】 赵喜娃；

【导师】 康山； 李琰；

【作者基本信息】 河北医科大学 ， 妇产科学， 2008， 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)是两种妇科常见病和多发病,是遗传因素和环境因素相互作用而引起的多基因遗传性疾病。内异症和腺肌病是引起育龄期妇女盆腔疼痛和不孕的重要原因,尽管有多种治疗方法,但效果并不令人满意,究其原因是这两种疾病的病因学和发病机理尚不完全明确。现在被普遍接受的理论为内异症是由于经血逆流所致,而腺肌病是由于子宫肌壁的防御屏障遭到破坏使子宫内膜得以侵入所致。然而无论子宫内膜细胞异位到子宫体以外还是子宫肌层,这种异位的内膜细胞想要存活并发展为内异症或腺肌病,一个前提条件是异位处细胞外基质(extracellular matrix, ECM)及基底膜(basement membrane, BM)的破坏和重建,而在这一过程中基质金属蛋白酶(matrix metalloproteinases,MMPs)起着重要的作用。在MMPs家族中,基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)因在组织中广泛的表达及其多重的功能而颇受关注,存在于MMP-2基因上的单核苷酸多态性( single nucleotide polymorphisms,SNPs)位点可能通过影响基因转录活性和蛋白表达水平而与一些疾病的发生发展相关。另外,金属蛋白酶组织抑制剂-2(tissue inhibitor of metalloproteinase-2,TIMP-2)是一种重要的内源性MMP-2抑制剂,其启动子区的SNPs也可能通过改变其转录活性而进一步影响TIMP-2和MMP-2的表达水平。因此,MMP-2和TIMP-2启动子区的一些SNPs可能通过改变MMP-2的表达水平进而影响内异症和腺肌病的发生发展。本研究旨在探讨MMP-2 -1306C/T和TIMP-2 -418G/C单核苷酸多态性与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,收集298例内异症患者、180例腺肌病患者和324例对照个体的静脉抗凝血各5ml,同时记录其病史及家族相关资料。采用蛋白酶K消化-饱和氯化钠盐析法提取外周血白细胞DNA,采用聚合酶链反应-限制性片段长度多态性方法(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)检测MMP-2和TIMP-2基因多态位点的基因型频率分布。数据统计分析采用SPSS11.5版软件包(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。比较各位点基因型频率的观察值与预期值并进行卡方检验行Hardy-Weinberg平衡分析。两组间的基因型分布比较采用行×列表χ2检验。以非条件Logistic回归法计算表示相对风险度的比值比(odds ratio, OR)及95%可信区间(confidence interval, CI)。采用似然比检验(likelihood ratio test)分析MMP-2和TIMP-2基因之间的交互作用。结果1内异症和腺肌病组患者的年龄、初潮年龄、孕次和产次与对照组相比,差异无统计学意义(P>0.05)。对照组中MMP-2和TIMP-2两个多态位点基因型频率分布均符合Hardy-weinberg平衡(P>0.05)。2 MMP-2基因启动子区-1306C/T多态C、T等位基因频率在内异症和对照组分别为88.9%、11.1%和87.0%、13.0%,两组相比无显著差异(P>0.05);C/C、C/T、T/T基因型频率分布在内异症(79.2%, 19.5%和1.3%)和对照组(75.7%, 22.8%和1.5%)间也无差异(P>0.05);与C/T+T/T基因型相比,C/C基因型不增加内异症的发病风险,OR值为1.23(95%CI=0.84~1.79)。3腺肌病组患者MMP-2 -1306C/T多态中C等位基因频率为92.2%,明显高于对照组(87.0%)(P<0.05);腺肌病患者组C/C、C/T、T/T三种基因型频率分别为85.0%、14.4%和0.6%,与正常对照组(75.7%、22.8%和1.5%)相比具有显著差异(P<0.05);与C/T+T/T基因型相比,C/C基因型明显增加腺肌病的发病风险,OR值为1.83(95%CI=1.13~2.96)。4 TIMP-2基因启动子区-418G/C多态G、C等位基因频率在内异症和对照组分别为83.7%、16.3%和81.3%、18.7%,两组相比无统计学意义(P>0.05);内异症组C/C纯合子的基因型频率(0.7%)明显低于对照组(3.7%)(P<0.05);与G/C+C/C基因型相比,G/G基因型并不能明显增加内异症的发病风险,OR值为1.08(95%CI=0.78~1.52)。5腺肌病组患者TIMP-2 -418G/C多态中G、C等位基因频率分别为82.2%和17.8%,与对照组(81.3%和18.7%)相比无统计学意义(P>0.05);腺肌病患者组G/G、G/C、C/C三种基因型频率分别为67.2%、30.0%和2.8%,与对照组(66.4%、29.9%和3.7%)相比亦无显著差异(P>0.05);与G/C+C/C基因型相比,G/G基因型不增加腺肌病的发病风险,OR值为1.04(95%CI=0.71~1.53)。6 MMP-2和TIMP-2基因的两个多态性位点不同基因型的联合分析结果显示,-1306C/C&-418G/G联合基因型在人群中最常见,在健康人群中占51.5% ;与-1306C/T+T/T&-418G/C+C/C联合基因型相比,其它3种基因型并不能明显增加内异症的发病风险,而-1306C/C&-418G/G和-1306C/C&-418G/C+C/C基因型能明显增加腺肌病的发病风险,OR值分别为2.37 (95%CI=1.05~5.35)和2.53(95%CI=1.08~5.95)。结论1 MMP-2基因启动子区-1306C/T单核苷酸多态可能与子宫内膜异位症的发病风险无关。2 MMP-2基因启动子区-1306C/T多态可能与子宫腺肌病的发病风险有关,即C/C基因型的携带者可能有较高的子宫腺肌病发病风险。3 TIMP-2基因启动子区-418G/C单核苷酸多态虽然未发现与子宫内膜异位症的发病风险相关,但内异症组C/C纯合子的频率明显低于对照组,可能是子宫内膜异位症发生的一个保护性因素。4未发现TIMP-2基因启动子区-418G/C单核苷酸多态与子宫腺肌病的发病存在关联。5 MMP-2和TIMP-2两个多态基因型联合分析发现,-1306C/C&-418G/G联合基因型在人群中最常见,在健康人群中占51.5%;与-1306C/T+T/T&-418G/C+C/C联合基因型相比,其它基因型并不能明显改变内异症的发病风险,而携带-1306C/C的联合基因型能明显增加腺肌病的发病风险。更多还原

【Abstract】 Objective:Endometriosis (EMs) and adenomyosis are common gynecological diseases, which are produced by multiple gene loci interacting with each other and with the environment. These two diseases can bring chronic pelvic pain and infertility for women of reproductive age. Although there are various methods to cure them, the result is unsatisfactory, and the reason due to the fact that aetiology and pathogenesis are not completely understood to date. It is the widely accepted that endometriosis is caused by the blood of menstruate countercurrented, while adenomyosis is caused by the decreased capacity of recovery for the muscular wall of the uterus. However, the endometrial cells implant either outside the uterine cavity or within the myometrial wall of the uterus, if implantation of these cells intends to be successful, one of the preconditions must be the degradation and turnover of extracellular matrix (ECM) and basement membrane (BM), in which the matrix metalloproteinases (MMPs) play important roles. MMP-2, of several members in the MMP family, is especially interesting because of its universal expression and multiple functions. The single nucleotide polymorphisms (SNPs) of MMP-2 may modify the transcriptional activity and protein expression of MMP-2, and further contribute to the development of some diseases. In addition, the tissue inhibitor of metalloproteinase-2 (TIMP-2) is the most important endogenous inhibitor of MMP-2. The SNPs of TIMP-2 promoter region may also modify the transcriptional activity and further influence protein expression of TIMP-2 and MMP-2. Thus, SNPs of MMP-2 and TIMP-2 promoter region may influence the risk of developing endometriosis and adenomyosis by modifying protein expression of MMP-2. The aim of the present study was to investigate association of MMP-2 -1306C/T and TIMP-2 -418G/C SNPs with the risk of endometriosis and adenomyosis.Methods:This case-contral study included 478 patients (298 with endometriosis and 180 with adenomyosis) and 324 frequency-matched healthy control women. Five milliliter of venous blood from each subject was drawn in Vacutainer tubes containing EDTA and stored at 4℃, while the information of every subject was obtained. The genomic DNA was extracted within one week after bleeding by using proteinase K digestion followed by a salting out procedure. Genotypes of the MMP-2 and TIMP-2 genes were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Statistical analysis was performed using SPSS11.5 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 by comparing the observed and expected genotype frequencies in study groups using Chi-square test. Comparison of the MMP-2 genotype and allelotype distribution in patients and healthy controls was performed by means of 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. The interactive influences of MMP-2 and TIMP-2 were analysed by likelihood ratio test.Results1 Age, menarche age, gravidity and parity among endometriosis, adenomyosis and control women had no significant difference (P>0.05). The genotype frequencies of MMP-2 and TIMP-2 in healthy controls did not significantly deviate from that expected for a Hardy-Weinberg equilibrium (P>0.05).2 The frequencies of the MMP-2 C and T allele among endometriosis patients and healthy controls were 88.9%, 11.1% and 87.0%, 13.0%, respectively; No significant difference in the MMP-2 allele distribution was shown between endometriosis patients and controls (P>0.05). The distribution of the C/C, C/T and T/T genotypes between endometriosis patients (79.2%, 19.5% and 1.3%, respectively) and controls (75.7%, 22.8% and 1.5%, respectively) also had no significant difference (P>0.05). Compared with the C/T+T/T genotypes, the C/C genotype could not increase the risk of developing endometriosis, the odds ratio was 1.23 (95%CI=0.84~1.79).3 The frequency of the MMP-2 C allele among adenomyosis patients (92.2%) was significantly higher than those in the healthy controls (87.0%) (P<0.05). The frequencies of the C/C, C/T and T/T genotypes among adenomyosis patients (85.0%, 14.4% and 0.6%, respectively) were significantly different from those in healthy controls (75.7%, 22.8% and 1.5%, respectively) (P<0.05). Compared with the C/T+T/T genotypes, the C/C genotype could significantly increase the risk of developing adenomyosis, the odds ratio was 1.83 (95%CI=1.13~2.96).4 The frequencies of the TIMP-2 G and C allele among endometriosis patients and healthy controls were 83.7%, 16.3% and 81.3%, 18.7%, respectively; No significant difference in the TIMP-2 allele distribution was shown between endometriosis patients and controls (P>0.05). The frequency of the C/C homozygote in endometriosis patients (0.7%) was significantly lower than that in the controls (3.7%) (P<0.05). Compared with the G/C+C/C genotypes, the G/G genotype could not increase the risk of developing endometriosis, the odds ratio was 1.08 (95%CI=0.78~1.52). 5 The frequencies of the TIMP-2 G and C allele among adenomyosis patients and healthy controls were 82.2%, 17.8% and 81.3%, 18.7%, respectively; There was no significant difference in the TIMP-2 allele distribution between adenomyosis patients and controls (P>0.05). The frequencies of the TIMP-2 G/G, G/C and C/C genotypes among adenomyosis patients and healthy controls were 67.2%, 30.0%, 2.8% and 66.4%, 29.9%, 3.7%, respectively; No significant difference in the TIMP-2 genotype distribution was shown between adenomyosis patients and controls (P>0.05). Compared with the G/C+C/C genotypes, the G/G genotype could not modify the risk of developing adenomyosis, the odds ratio was 1.04 (95%CI=0.71~1.53).6 The combined effect of MMP-2 -1306C/T and TIMP-2 -418G/C genotypes showed that the -1306C/C&-418G/G was the most frequent combined genotype in the population, which was 51.5% in controls. Compared with the -1306C/T+T/T&-418G/C+C/C genotype, the other combined genotypes could not modify the risk of developing endometriosis; However, the -1306C/C&-418G/G and -1306C/C&-418G/C+C/C genotypes could significantly increase the risk of developing adenomyosis, the odds ratio were 2.37 (95%CI=1.05~5.35) and 2.53 (95%CI=1.08~5.95), respectively.Conclusions1 The MMP-2 -1306C/T SNP might not be related to the risk of endometriosis development. The MMP-2 C/C genotype did not significantly increase the risk of the endometriosis development compared to C/T+T/T genotypes.2 The MMP-2 -1306C/T SNP was associated with the risk of developing adenomyosis. The subjects carrying C/C genotype could be associated with the increased risk of developing adenomyosis.3 Although no association of the TIMP-2 -418G/C SNP with the risk of endometriosis development was showed, the frequency of the C/C homozygote in endometriosis patients was significantly lower than that in the controls, which might be a protective factor for the development of endometriosis.4 There was no significant association between the TIMP-2 -418G/C SNP and the risk of developing adenomyosis.5 The combined effect of MMP-2 and TIMP-2 genotypes indicated that the -1306C/C&-418G/G was the most frequent combined genotype in the population, which was 51.5% in controls. Compared with the -1306C/T+T/T&-418G/C+C/C genotype, although the other genotypes could not modify the risk of developing endometriosis, the -1306C/C&-418G/G and -1306C/C&-418G/C+C/C combined genotypes could significantly increase the risk of developing adenomyosis更多还原