【作者】 连艳；

【导师】 杨鹰；

【作者基本信息】 第三军医大学 ， 妇产科学， 2008， 硕士

【摘要】 背景和目的目前绝经期综合征多采用激素替代疗法(Hormone Replacement Therapy,HRT),但长期使用非对抗性雌激素易引发子宫内膜增生,增加子宫内膜腺癌的危险性。另外绝经期雌激素水平下降还可造成女性泌尿生殖道支撑组织萎缩和张力减退,引发压力性尿失禁(Stress urinary incontinence,SUI),而雌激素替代治疗SUI的疗效不甚理想,有学者假设可能与雌激素受体(Estrogen recepter,ER)亚型比例变化相关。子宫、阴道组织中ERα、ERβ分布及含量改变可以引起雌激素类药物作用效应的差异。目前关于绝经期子宫、阴道组织中ERα、ERβ的分布及含量变化,两种受体亚型介导雌激素生物效应的差异及相互作用,国内外的研究结果不相一致。本研究采用免疫组化、免疫荧光双标(DLTI)激光扫描共聚焦显微镜(LSCM)及半定量RT-PCR技术,观察成年SD雌鼠、去势及药物干预后大鼠子宫、阴道ER亚型的分布及共表达状况,初步探讨ER亚型及ERα/ERβ值的变化与子宫内膜增殖、阴道萎缩之间的关系,为研究外源性雌激素对子宫、阴道ER亚型的调节及绝经后妇女合理补充雌激素提供理论和实验依据。主要方法和结果1. 80只4月龄体重320～350g健康雌性SD大鼠随机分为正常组、假手术组、去势组、去势+戊酸雌二醇组(药物组),每组20只。正常组未经干预,假手术组打开腹腔但不切除卵巢,药物组于术后4周用戊酸雌二醇生理盐水混悬液连续灌胃8周后处死大鼠。观察大鼠子宫、阴道大体形态学改变和组织学改变,并检测血中雌二醇和卵泡刺激素水平。结果:去势组于术后连续5天阴道上皮涂片染色无规律的动情期改变。去势组子宫、阴道明显萎缩,体积减小,药物组子宫、阴道湿重显著增加。HE染色去势组子宫内膜全层萎缩,腺体稀疏;阴道壁变薄,黏膜上皮萎缩。予雌激素后,子宫内膜明显增厚,腺体密集,偶可见腺腔囊状扩张;阴道壁轻度增厚,出现特征性皱褶结构。去势组大鼠血清雌二醇浓度明显降低、FSH水平明显升高,药物组E2水平较正常组、假手术组高。2.免疫组化SP法检测ERα、ERβ蛋白;应用免疫荧光组织化学双标记染色技术,在激光共聚焦扫描显微镜下观察ERα、ERβ的表达情况;半定量RT-PCR检测ERα、ERβmRNA的表达水平。结果:1)去势组子宫ERα蛋白表达均较正常组和假手术组明显减弱,ERβ几乎未见表达,ERα/ERβ值升高。药物组ERα蛋白的表达较去势组、正常组和假手术组明显增强,ERβ表达无显著改变,ERα/ERβ值显著升高。去势组ER mRNA表达降低,以ERβ降低更显著。药物组ERαmRNA表达较去势组明显增强,甚至高于正常组。2)去势组阴道ERα蛋白表达水平较正常组、假手术组降低,ERβ表达水平下降更为明显,ERα/ERβ值升高。药物组鳞状上皮细胞ERα蛋白表达较去势组明显增强,基质纤维细胞和平滑肌细胞ERβ表达无改变,ERα/ERβ值显著升高。去势组ER mRNA表达均降低,以ERβ表达下降更为显著,药物组ERαmRNA表达较去势组明显增强。结论:1.从组织形态学和功能上均证实去势大鼠模型的成功建立。2.去势大鼠子宫ERα、ERβmRNA及蛋白的表达均下降,以ERβmRNA和蛋白的表达下降更显著,ERα/ERβ值升高。戊酸雌二醇明显上调去势大鼠子宫ERαmRNA和蛋白的表达,对ERβmRNA和蛋白的表达无明显影响,ERα/ERβ值进一步升高。3.去势引起的雌激素水平下降,造成阴道组织三种细胞ER亚型表达的下降或缺失,ERβmRNA和蛋白的表达下降更显著,ERα/ERβ值升高。雌激素替代治疗明显上调鳞状上皮细胞ERα蛋白的表达;ERT对基质纤维细胞和平滑肌细胞ERβ蛋白的表达无影响。更多还原

【Abstract】 Background and objectiveIt is well-known that hormone replacement herapy(HRT) has become the principal therapic method in women with perimenopausal syndrome. However, long-term use of unopposed estrogens would induce endometrial hyperplasia and increase risk of adenocarcinoma endometrium. In addition, Decreased level of estrogen could lead to telatrophy and hypotonia of female genitourinary tract, and initiate stress urinary incontinence(SUI), but the therapeutic outcome with estrogen is not effective enough, so some researchers hypothesized that SUI might correlate with estrogen receptor. Changes of distribution and content of estrogen receptor alpha(ERα) and estrogen receptor beta(ERβ) could affect drug action of the estrogen. The research results were different about biological effect and interaction of the two subtypes.In present study, we evaluated distribution and coexpression of ERα, ERβin uretus and vagina in adult male SD rats subjected to ovariedctomy and intervention of Estradiol Valerate after ovariedctomy, investigated relationship between changes of ER subtypes, ERα/ERβand endometrial hyperplasia and vaginal atrophy, provide theoretical basis for researching regulation of exogenous estrogen to ER subtypes of uretus and vagina, and appropriate supplying estrogen of postmenopause.Main Methods and results1. A total number of 80 female SD rats aged 4 months weighing about 340g each were randomly assigned into 4 groups: control group (n=20), sham-operated group (n=20), ovariectomied group (n=20) and drug group (n=20). The drug group was treated estradiol valerate everyday by intragastric administration after 4 weeks of Ovariectomy. The other three groups were medicated aequales normal saline by intragastric administration. 8 weeks later, all the rats were killed and the tissues of uterus and vagina and blood sample was collected. Morphological and histopathological changes of uretus and vagina were observed. Levels of estradiol and follicle stimulating hormone(FSH) in plasma were measured. The results showed that there were no estruation changes of vaginal epithelium detected in consecutive 5 days after ovariectomy. Uretus and vagina tissues became athophy, but they return to nomal after drug-taking. HE staining illustrated that endomembrance became atrophy and grand become rarefaction in ovariectomied group, and became thicker after drug-taking. Estradiol Level in plasma decreaed and plasma FSH content elevated significantly. Plasm Estradiol level rised and FSH concentration declined after taking estradiol valerate.2. Immunohistochemistry method and Double-labelling technique of immunofluorescence(DLTI) +Laser scanning confocal microscopy(LSCM) was used to detect the expression and coexpression of ER-αand ER-βin superficial and deep compartments of the Uterus and vagina. The levels of mRNA gene expression of the two subtypes of estrogen receptors were measured by semi-quantitative RT-PCR. (1)The findings demonstrated that estrogen receptor alpha and beta were expressed in luminal epithelial cells, glandular epithelial cells, stromal fibroblast, vascular endothelial cells and the cell nucleus and cytoplasm of smooth muscle cells, especially in luminal epithelial cells and glandular epithelial cells. The Expression of ER alpha is significantly higher than that of ER beta. In terms of the expression of ER alpha, which in the ovariectomied group was lower than that of the control group and sham-operated group and the ratios of ER-α/ER-βis risen. However, there are nearly no expression of the ER beta in the ovariectomied group. The Expression of estrogen receptor alpha in the Uterus was significantly upregulated in drug group, whereas estrogen receptor beta was not upregulated significantly.(2)Estrogen receptor alpha and beta were expressed in squamous epithelial cells, stromal fibroblast, vascular endothelial cells, smooth muscle cells, especially in squamous epithelial cells. The Expression of ER alpha is significantly stronger than that of ER beta. Lower expression of ER isoforms, especially ER beta in ovariectomied group was showed.The Expression of estrogen receptor alpha in the vagina was significantly upregulated in drug group, whereas estrogen receptor beta was not significant up regulated.Conclusions1. successfully establishing animal model of Ovariectomy and drug intervention after Ovariectomy.2. ER-αis a major subtype of Estrogen receptor expressed in uterus of rats. It was demonstrated that the expression of ER was shown lower after ovariectomy, especially that of ER-βand the ratios of ER-α/ER-βis risen. It suggested that estradiol valerate could upregulated expression of ER-αbut not contribution to ER-β.3. ER-αis major expression isoforms of Estrogen receptor in vaginal tissue of rats. It was demonstrated down-regulation of ER-αand ER-βexpression in the vaginal tissues after ovariectomy, especially that of ER-βand the ratios of ER-α/ER-βis risen.The findings showed that estradiol valerate could upregulated expression of ER-αbut ER-β.更多还原