Twist异常甲基化介导的细胞迁移和侵袭在子宫内膜异位症发病机制中的作用

发布时间：2018-06-18 04:50

本文选题：子宫内膜异位症 + Twist　；参考：《浙江大学》2017年博士论文

【摘要】：子宫内膜异位症(endometriosis,EMs)是指具有生长功能的子宫内膜组织(腺体和间质)出现在子宫腔被覆内膜及宫体肌层以外的其他部位。它是育龄妇女的常见病,以25～45岁妇女多见,发病率高达10%-15%,近年有明显上升趋势,它所引起的慢性盆腔疼痛、痛经和不孕严重影响了妇女健康和生活质量。自1860年Von Rokitansky首先描述子宫内膜异位症以来,本病发病机制至今仍未完全阐明。虽然Sampson等提出的经血逆流学说被广为接受,但经血逆流在生育期妇女中非常普遍,发生率高达76%～90%,而EMs的发病率却仅占10%～15%,说明经血逆流可能只是诱因,而异位子宫内膜的迁移和侵袭能力以及对卵巢等盆腔器官和腹膜的粘附、浸润等生物学行为,才可能是其发病的关键。近年来,有文献报道证实了表观遗传学为子宫内膜异位症发病的重要机制之一,基因甲基化是表观遗传学的一个重要组成部分。Twist是新近发现的凋亡抑制蛋白,作为基因组中的高度保守序列,在胚胎发育和人类疾病发展过程中普遍存在着甲基化。该基因的功能包括参与上皮-间质转化促进细胞迁移侵袭、抗细胞凋亡、促进肿瘤血管生成和导致染色体不稳定等。一般来说基因甲基化可使基因表达沉默,而去甲基化则可使其表达增加。但迄今为止,Twist基因启动子的甲基化状态与子宫内膜异位症的关系国内外尚未见报道。Twist可通过抑制E-cadherin和诱导N-cadherin来调节上皮-间质转化(epithelial-mesenchymal transition,EMT),是上皮细胞获得迁移能力并侵犯到其他部位的有效方式,在肿瘤的发生及浸润过程中扮演了重要角色,同时也可能是具有恶性生物学特征的EMs重要发病机制之一。本研究的目的是检测和比较Twist、E-cadherin和N-cadherin在卵巢型异位内膜及其在位子宫内膜、非EMs者子宫内膜组织和细胞中的表达差异,研究Twist对EMs在位内膜间质细胞迁移和侵袭能力的影响,研究三种不同组织中Twist甲基化状态的差异,探讨Twist异常甲基化所介导的细胞迁移和侵袭在EMs发病机制中可能的作用,同时为EMs靶向药物治疗和复发的预防提供新的思路。第一部分 Twist、N-cadherin和E-cadherin在不同子宫内膜组织中的表达差异目的:在翻译和转录水平上,研究Twist、N-cadherin和E-cadherin在卵巢型异位内膜、EMs在位内膜和非EMs子宫内膜组织中的表达差异。方法:用Western blot和免疫组织化学法检测Twist、N-cadherin和E-cadherin蛋白,比较卵巢型异位内膜、在位内膜与非EMs子宫内膜组织三种蛋白的表达和定位情况;用定量RT-PCR检测并比较三组组织中Twist、N-cadherin和E-cadherin的mRNA表达情况。结果:1.免疫组化结果显示Twist、N-cadherin和E-cadherin在子宫内膜腺上皮和间质细胞均有表达,Twist和N-cadherin的表达:卵巢型异位内膜在位内膜非EMs子宫内膜,E-cadherin则相反,组间差异均有显著性(P0.05)。Spearman分析表明N-cadherin和Twist呈正相关,E-cadherin与Twist呈负相关。2.Western blot提示Twist和N-cadherin蛋白表达量由高到低排列顺序为:卵巢型异位内膜在位内膜非EMs子宫内膜,且差异有显著性(P0.05)。E-cadherin蛋白的表达:非EMs子宫内膜EM在位内膜卵巢型异位内膜,差异有统计学意义(P0.05)。3.RT-PCR显示Twist和N-cadherinmRNA表达量在卵巢型异位内膜和在位内膜的表达较非EMs子宫内膜明显升高(P0.05);E-cadherin mRNA表达在非EMs子宫内膜中表徶最高,组间差异有显著性。结论:Twist与EMs的发病有关,可能通过介导上皮间质转化参与EMs的发病。第二部分高表达的Twist促进子宫内膜间质细胞迁移和侵袭能力目的:探讨Twist对在位子宫内膜间质细胞迁移和侵袭能力的影响。方法:原代培养子宫内膜间质细胞,构建质粒过表达载体(cmv-mcs-3flag-sv40-Neom-Twist)并稳定转染至在位子宫内膜间质细胞,使其过表达Twist;用Western blot检测转染前后在位子宫内膜间质细胞的Twist、N-cadherin、E-cadherin蛋白表达情况;用RT-PCR检测比较转染前后在位子宫内膜间质细胞Twist、N-cadherin、E-cadherin的mRNA表达情况;Transwell法检测子宫内膜间质细胞迁移和侵袭能力的改变。免疫荧光法检测卵巢型异位内膜、在位子宫内膜和非EMs子宫内膜的间质细胞形态和三种蛋白的表达差异以及在细胞内的定位情况。结果:1.成功转染Twist至在位子宫内膜间质细胞引起过表达后,Western blot和RT-PCR检测发现N-cadherin在蛋白和mRNA水平表达增加,E-cadherin蛋白和mRNA表达则下降,转染前后差异有统计学意义。2.Transwell法检测发现,在位子宫内膜间质细胞转染Twist后细胞迁移和侵袭能力均明显增加,差异有显著性。3.免疫荧光检测发现三组子宫内膜间质细胞均表达Twist、N-cadherin和E-cadherin,Twist和N-cadherin在卵巢型内膜细胞表达最明显,E-cadherin在非EMs子宫内膜细胞表徶最明显,异位子宫内膜间质细胞表型更为多态性。结论:Twist过表达后在位子宫内膜间质细胞迁移和侵袭能力增加,同时上调N-cadherin的蛋白和mRNA表达,下调E-cadherin的蛋白和mRNA表达,提示Twist引起EMs发病的机理可能与上皮间质转化引起的迁移和侵袭能力增加有关。第三部分 Twist基因启动子甲基化在不同子宫内膜组织中的差异目的:探讨卵巢型异位内膜、在位内膜和非EMs子宫内膜组织Twist基因启动子甲基化状态的差异。方法:设计Twist基因启动子CpG岛的6对引物后,采用焦磷酸测序法的甲基化特异性PCR(MSP),检测三组内膜组织中启动子甲基化水平的差异。结果:Twist基因启动子在卵巢型异位内膜和在位子宫内膜的某些区域呈低甲基化状态。结论:推测卵巢型异位内膜和在位子宫内膜组织Twist基因启动子的区域性低甲基化状态,可引起Twist蛋白过表达,从而可能直接导致EMs的发病。
[Abstract]:Endometriosis (endometriosis, EMs) refers to the appearance of endometrium with growth function (glands and stroma) in other parts of the endometrium outside the endometrium and the intrauterine layer. It is a common disease of women of childbearing age. It is a common disease of women of childbearing age. The incidence of the 25~45 year old women is more common and the incidence is up to 10%-15%. Pelvic pain, dysmenorrhea and infertility seriously affect the health and quality of life of women. Since the first description of endometriosis in Von Rokitansky in 1860, the pathogenesis of this disease has still not been fully elucidated. Although the theory of blood reflux proposed by Sampson is widely accepted, the blood reverse flow is very common among women in the childbearing period, and the incidence is high. From 76% to 90%, the incidence of EMs is only 10% to 15%, indicating that the transmigration and invasion of the ectopic endometrium, as well as the adhesion to the pelvic organs and peritoneum of the ovary, and the infiltration of the peritoneum, may be the key to the disease. In recent years, the literature has reported that epigenetics is in the uterus. One of the important mechanisms of the pathogenesis of membrane heterotopic, gene methylation is an important part of epigenetics,.Twist is a newly discovered inhibitor of apoptosis. As a highly conservative sequence in the genome, methylation exists in the development of embryo and human disease. The function of this gene includes participation in epithelial mesenchymal transition. It promotes cell migration and invasion, anti apoptosis, promotes tumor angiogenesis and causes chromosomal instability. Generally, gene methylation can make gene expression silent, while demethylation can increase its expression. However, the relationship between the methylation status of the Twist gene promoter and endometriosis has not yet been reported to the home and abroad. .Twist can regulate epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) by inhibiting E-cadherin and inducing N-cadherin. It is an effective way for epithelial cells to obtain migration and invasion to other parts. It plays an important role in the process of tumor development and invasion, and may also have malignant biological characteristics. The purpose of this study is to detect and compare the differences in the expression of Twist, E-cadherin and N-cadherin in the ovarian endometrium and its eutopic endometrium, in the endometrium and in the non EMs endometrium, and to study the effect of Twist on the migration and invasion of the eutopic interstitial cells of the endometrium in EMs, and the study of three different tissues. The difference in Twist methylation status, to explore the possible role of Twist abnormal methylation mediated cell migration and invasion in the pathogenesis of EMs, and to provide new ideas for the prevention of EMs targeting drug therapy and recurrence. Part I, Twist, N-cadherin and E-cadherin expression differences in different endometrium tissues: in translation and At transcriptional level, the differences in expression of Twist, N-cadherin and E-cadherin in ovarian ectopic endometrium, EMs eutopic endometrium and non EMs endometrium were studied. Methods: Western blot and immunohistochemistry were used to detect Twist, N-cadherin and E-cadherin proteins, ovarian endometrium, eutopic endometrium and non EMs endometrium, three species. The expression and localization of protein were detected by quantitative RT-PCR and the mRNA expression of Twist, N-cadherin and E-cadherin in the three groups was compared. Results: 1. immunohistochemical results showed that Twist, N-cadherin and E-cadherin were expressed in the endometrium and interstitial cells of the endometrium, and the expression of Twist and N-cadherin: ovarian ectopic endometrium in the position of the endometrium The membrane was not EMs endometrium, and E-cadherin was opposite, and there was a significant difference between the groups (P0.05).Spearman analysis showed that N-cadherin and Twist were positively correlated, E-cadherin and Twist showed a negative correlation of.2.Western blot suggesting Twist and N-cadherin protein expression from high to low order: the ectopic endometrium in the ovum type endometrium was not the endometrial endometrium, and the difference was poor. The expression of P0.05.E-cadherin protein: the non EMs endometrium EM eutopic endometrium endometrium, the difference was statistically significant (P0.05).3.RT-PCR showed that the expression of Twist and N-cadherinmRNA in the ovarian endometrium and the eutopic endometrium was significantly higher than that of the non EMs endometrium (P0.05); E-cadherin mRNA was expressed in non ovarian endometrium. Conclusion: Twist is associated with the pathogenesis of EMs and may be involved in the pathogenesis of EMs by mediating epithelial transformation. The second part of high expression Twist promotes the migration and invasion of endometrial stromal cells: To explore the ability of Twist to migrate and invasiveness of endometrial stromal cells. Methods: the primary endometrial stromal cells were cultured, the plasmid overexpression vector (cmv-mcs-3flag-sv40-Neom-Twist) was constructed and transfected to the eutopic endometrial stromal cells to overexpress Twist. The expression of Twist, N-cadherin, E-cadherin protein in the eutopic endometrium cells before and after transfection was detected by Western blot; and RT-PC was used in RT-PC. R was used to detect the mRNA expression of Twist, N-cadherin, E-cadherin in eutopic endometrial stromal cells before and after transfection. Transwell assay was used to detect the change of migration and invasion of endometrial stromal cells. Immunofluorescence was used to detect ectopic endometrium, the morphology of interstitial cells in the eutopic endometrium and non EMs endometrium and the table of three proteins Results: after 1. successful transfection of Twist to eutopic endometrial stromal cells, the expression of N-cadherin at the level of protein and mRNA increased, the expression of E-cadherin protein and mRNA decreased, and the difference between before and after transfection was statistically significant. The cell migration and invasion ability of endometrial stromal cells transfected with Twist were increased obviously. The difference was significant.3. immunofluorescence detection found that three groups of endometrial stromal cells expressed Twist, N-cadherin and E-cadherin, Twist and N-cadherin were most obvious in ovarian endometrial cells, E-cadherin in non EMs endometrium. The phenotype of ectopic endometrium was more polymorphic. Conclusion: after Twist overexpression, the migration and invasion ability of eutopic endometrial stromal cells increased, and the expression of protein and mRNA was up-regulated, the protein and mRNA expression of E-cadherin was down regulated, suggesting that the mechanism of Twist to cause the pathogenesis of EMs may be related to the transformation of epithelial mesenchymal transition. Increased migration and invasiveness. Third differences in the methylation of the Twist gene promoter in different endometrium purposes: To explore the difference in the methylation status of the Twist gene promoter in the ovarian ectopic endometrium, the eutopic endometrium and the non EMs endometrium. Methods: after the design of 6 pairs of primers in CpG island of the promoter of the Twist gene, The methylation specific PCR (MSP) of the pyrosequencing method was used to detect the difference in the level of promoter methylation in the three groups of endometrium. Results: the Twist gene promoter was methylation in some regions of the ovarian ectopic endometrium and in the eutopic endometrium. Conclusion: the Twist gene of the ovarian endometrium and the eutopic endometrium was presumed to be initiated. The regional hypomethylation of mover can cause over expression of Twist protein, which may directly lead to the pathogenesis of EMs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R711.71

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