【作者】 廉红梅；

【导师】 刘义；

【作者基本信息】 华中科技大学 ， 妇产科学， 2008， 硕士

【摘要】 背景和目的:子宫内膜异位症(endometriosis , EM)是子宫内膜碎片通过“异地”黏附、侵袭、生长种植于子宫腔以外部位的雌激素依赖性疾病,在位与异位子宫内膜细胞主要由上皮细胞和间质细胞组成,因此在EM的研究中,获得纯化的内膜细胞是很重要的一个环节。本研究旨在建立人子宫正常内膜、子宫内膜异位症(内异症)在位及异位子宫内膜间质细胞的分离、培养的方法,为进一步探讨子宫内膜异位症发生,发展的分子生物学机制提供一个理想的实验模型。方法:12例正常子宫内膜、9例在位子宫内膜及7例异位子宫内膜经分段酶解、筛网过滤、沉降及贴壁纯化技术进行分离、纯化和体外培养,光镜观察,应用鼠抗人波形蛋白单克隆抗体及鼠抗人细胞角蛋白单克隆抗体并采用SP法,对间质细胞进行免疫细胞化学染色鉴定。结果:10例正常子宫内膜、6例在位子宫内膜及4例异位子宫内膜标本分离、培养成功,间质细胞纯度均可达93%以上,并均可传代6～8代。结论:采用分段酶解、筛网过滤、沉降及贴壁纯化的方法可获得纯度较高的子宫内膜间质细胞,为进一步研究内异症发生、发展的分子生物学机制提供一个理想的实验模型。目的:研究17β-雌二醇(17β-E2)对子宫内膜异位症(内异症)患者在位子宫内膜间质细胞趋化因子SDF-1和其受体CXCR4mRNA及蛋白表达的影响,探讨SDF-1/CXCR4在介导雌激素促进内异症发生发展中的作用。方法:体外分离、培养内异症患者在位子宫内膜间质细胞。用不同浓度17β-E2(10-12、10-10、10-8、10-6 mol/L)处理子宫内膜间质细胞12,24和48h,利用实时定量PCR(RQ-PCR)技术和流式细胞检测技术分别检测17β-E2处理前后子宫内膜间质细胞趋化因子SDF-1及其受体CXCR4 mRNA及蛋白的表达;同法分析雌激素受体(ER)拮抗剂ICI182780(10-8mol/L)对17β-E2促进SDF-1/CXCR4 mRNA和蛋白表达的影响。结果:不同浓度17β-E2作用于内异症患者在位子宫内膜间质细胞12、24、48h后,SDF-1及其受体CXCR4 mRNA及蛋白的表达呈不同程度增高,于10-10 mol/L作用最明显。雌激素受体拮抗剂ICI182780能明显抑制17β-E2对子宫内膜间质细胞SDF-1/CXCR4 mRNA和蛋白的表达。结论:雌激素可促进内异症患者在位子宫内膜间质细胞SDF-1/CXCR4 mRNA及蛋白的表达,SDF-1/CXCR4可能参与了内异症的形成和发展。更多还原

【Abstract】 Objective To explore the method of culture for human stromal cell of normal endometrium, eutopic endometrium and ectopic endometrium.Methods Enzymolyses,filtration ,sedimentation and pasted wall purification were used to isolate and culture the stromal cells of twelve samples of normal endometrium, nine sampls of eutopic endometrium and six samples of ectopic endometrium. These stromal cells were observed by light microscopy and identified by immunocytochemical staining uising mouse antihuman cytokeratin and vimentin antibody.Results Ten normal endometrial sampls, six eutopic endometrial sampls and four ectopic endometrial sampls are isolated and cultured successfully. The purity of stromal cells is 93% verified by cytokeratin and vimentin antibody staining, all of these tromel cells could be transfered for six to eight generations.Conclusion Highly purified stromal cells can be obtained through enzymolysis filtration, sedimentation and pasted wall ,purification, the cellular model will be helpful to investigate the pathogenesis of endometriosis and therapy of endometriosis. Objective:To investigate the effect of 17β-estradiol on expression of SDF-1/CXCR4 mRNA and protein by endometrial stromal cells of endometriosis and the role of SDF-1/CXCR4 in pathogenesis of endometriosis.Methods:Separate and culture the primary eutopic ESC of endometriosis in vitro, The cells were treated by 17β-estradiol (17β-E2 )with various concentrations(0、10-12、10-10、10-8、10-6 mol/L)for different time(0, 12, 24, 48h). The expressions of SDF-1/CXCR4 mRNA and protein in ESC of endometriosis were analyzed by Real-time quantitative PCR(RQ-PCR) and Flow Cytometry. Meanwhile, the expression of SDF-1/CXCR4 mRNA in cells by the ER inhibitor ICI182780(10-8 mol/L) and 17β-E2 treated were detected in the same waysResults:After treating of 17β-estradiol in different concentration for 12h、24h and 48h, the expression of SDF-1/CXCR4 mRNA and protein are both up-regulated to different extents , the role of 17β-estradiol is in evidence in concentration of 10-10mol/L. Theses kinds of roles of 17β-estradiol on SDF-1 and CXCR4ER can be blocked by ER inhibitor ICI182780 (10-8 mol/L) obviously.Conclusion:The expression of SDF-1/CXCR4 mRNA and protein can be promoted by 17β-estradiol in eutopic ESC of endometriosis. SDF-1 and its receptor CXCR4 may have correlation with the development of endometriosis.更多还原