巨噬细胞及其诱导的上皮间质转化在子宫腺肌病发病机制中作用的研究

发布时间：2018-05-24 13:44

本文选题：子宫腺肌病 + 上皮间质转化(EMT)　；参考：《山东大学》2017年硕士论文

【摘要】：背景:子宫腺肌病是育龄期及围绝经期女性常见的良性非瘤性的的疾病。其生物学行为类似于恶性肿瘤。近年来,研究证明M2巨噬细胞诱导的上皮间质转化(EMT)在肿瘤进展中作用显著。而先前的部分研究显示巨噬细胞聚集在腺肌症异位病灶中,另有部分研究显示腺肌症病灶中有上皮间质转化(EMT)的现象。因此,我们推测巨噬细胞及其诱导的上皮间质转化(EMT)在腺肌症发病过程中可能具有重要的意义。第一部分巨噬细胞与子宫腺肌病病灶中上皮间质转化现象关系密切研究目的:1.验证子宫腺肌症异位病灶中存在上皮间质转化(EMT)的现象。2.检测巨噬细胞在子宫腺肌症异位病灶中的分布。3.观察子宫腺肌症异位病灶中上皮间质转化(EMT)与巨噬细胞分布之间的关系。研究方法:使用免疫组织化学染色法和免疫荧光法对收集的正常子宫内膜、子宫腺肌症在位内膜和异位内膜临床样本进行染色,检测E-cadherin,N-cadherin,CK7,vimentin,S100A4和CD68在标本中的表达情况,分析异位病灶中上皮间质转化的比例,同时分析巨噬细胞的分布密度与上皮间质转化相关分子表达量之间的关系。研究结果:1.子宫腺肌症异位病灶中存在上皮间质转化(EMT)的现象,且比例较高。2.子宫在腺肌症异位病灶及在位内膜中有大量的巨噬细胞浸润,其分布与月经周期无关。3.在子宫腺肌症异位腺体周围,巨噬细胞的分布密度同上皮细胞中上皮性标志物表达呈负相关,同间质标志物表达呈正相关。结论:巨噬细胞浸润与子宫腺肌病病灶中的上皮间质转化(EMT)现象关系密切。巨噬细胞是腺肌症异位病灶发生上皮间质转化(EMT)的重要参与者。第二部分巨噬细胞诱导子宫内膜上皮细胞发生上皮间质转化(EMT)研究目的:1.探讨巨噬细胞能否诱导Ishikawa细胞发生上皮间质转化(EMT)。2.探讨巨噬细胞能否诱导子宫腺肌症在位内膜及正常子宫内膜发生上皮间质转化(EMT)。研究方法:将巨噬细胞与Ishikawa细胞、腺肌症在位内膜上皮细胞、正常子宫内膜上皮细胞共培养。蛋白质印迹法(Western Blot)检测共培养前后E-cadherin,N-cadherin,CK7,vimentin,S100A4 在 Ishikawa 细胞、腺肌症在位内膜上皮细胞、正常子宫内膜上皮细胞中的表达。通过细胞侵袭实验检测Ishikawa细胞与巨噬细胞共培养发生上皮间质转化(EMT)后的侵袭能力的改变。通过普通光学显微镜和透射电镜(TEM)检测观察Ishikawa细胞与巨噬细胞共培养发生上皮间质转化(EMT)后的细胞形态及细胞间连接的改变。通过流式细胞术(FCM)检测Ishikawa细胞与巨噬细胞共培养发生上皮间质转化(EMT)后的细胞增殖和凋亡改变。最后通过基因芯片表达分析来检测Ishikawa细胞与巨噬细胞共培养发生上皮间质转化(EMT)过程中信号通路的变化。研究结果:1.巨噬细胞诱导Ishikawa细胞发生上皮间质转化(EMT)。2.巨噬细胞诱导子宫腺肌症在位内膜及正常子宫内膜上皮细胞发生上皮间质转化(EMT)。3.子宫腺肌症在位内膜与正常子宫内膜相比发生上皮间质转化(EMT)的潜力更显著。结论:巨噬细胞可以诱导子宫内膜上皮细胞发生上皮间质转化(EMT)。与正常的子宫内膜相比,腺肌症在位内膜在与巨噬细胞共培养后发生上皮间质转化(EMT)更具潜力。第三部分共培养体系中上皮细胞诱导巨噬细胞向M2极化研究目的:1.探讨Ishikawa细胞能否诱导巨噬细胞向M2极化。2.探讨子宫腺肌症在位内膜及正常子宫内膜能否诱导巨噬细胞向M2极化。研究方法:通过流式细胞术(FCM)和聚合酶链式反应(PCR)检测与Ishikawa细胞、腺肌症在位内膜上皮细胞、正常子宫内膜上皮细胞共培养前后的巨噬细胞中CD163,IL-10,MMP12 的表达。研究结果:1.Ishikawa细胞诱导巨噬细胞向M2极化。2.子宫腺肌症在位内膜及正常子宫内膜均能诱导巨噬细胞向M2极化。3.与腺肌症在位内膜相比,正常子宫内膜上皮细胞诱导巨噬细胞向M2极化作用更明显。结论:Ishikawa细胞,子宫腺肌症在位内膜及正常子宫内膜上皮细胞均能诱导巨噬细胞向M2型转化。同时,正常子宫内膜上皮细胞诱导巨噬细胞向M2极化作用明显强于腺肌症在位子宫内膜上皮细胞。
[Abstract]:Background: adenomyosis is a common benign non tumor disease of women at the childbearing age and perimenopausal period. Its biological behavior is similar to that of malignant tumor. In recent years, studies have shown that M2 macrophage induced epithelial transformation (EMT) plays a significant role in the progression of tumor. Another part of the study showed the phenomenon of epithelial mesenchymal transition (EMT) in adenomyosis. Therefore, we speculate that macrophages and their induced epithelial transformation (EMT) may have important significance in the pathogenesis of adenomyosis. The first part of the macrophage is closely related to the epithelial mesenchymal transition in the lesions of the adenomyosis. Objective: 1. to verify the existence of epithelial mesenchymal transformation (EMT) in ectopic lesions of the adenomyosis (.2.); the distribution of macrophages in ectopic lesions of the adenomyosis (.3.) observation of the relationship between epithelial mesenchymal transition (EMT) and the distribution of macrophages in ectopic lesions of the uterus. Method of immunohistochemical staining: immunohistochemical staining The expression of E-cadherin, N-cadherin, CK7, vimentin, S100A4 and CD68 in the specimens of the endometrium and ectopic endometrium in the normal endometrium and adenomyosis of the uterus were stained with the immunofluorescence method, and the proportion of the epithelial mesenchymal transition in the ectopic lesion was analyzed, and the distribution density of macrophages and the epithelium were analyzed. The relationship between the expression of mass transformation related molecules. Results: 1. the phenomenon of epithelial mesenchymal transformation (EMT) exists in ectopic lesions of the adenomyosis, and a high proportion of.2. uterus has a large amount of macrophage infiltration in the ectopic and eutopic endometriosis of the uterus, and the distribution of the uterus is not related to the menstrual cycle of.3. around the adenomyosis of the uterus. The distribution density of macrophages is negatively correlated with the expression of epithelial markers in epithelial cells, and there is a positive correlation with the expression of interstitial markers. Conclusion: macrophage infiltration is closely related to epithelial mesenchymal transition (EMT) in adenomyosis lesions. Macrophage is an important participant in epithelial mesenchymal transformation (EMT) in adenomyosis ectopic focus. Second the study of epithelial mesenchymal transformation (EMT) in endometrial epithelial cells induced by macrophages: 1. to explore whether macrophages can induce epithelial mesenchymal transition in Ishikawa cells (EMT).2. to explore whether macrophages can induce epithelial mesenchymal transition (EMT) in the eutopic endometrium and normal Subendometrium of the uterus (EMT). Macrophages and Ishikawa cells, adenopathy in eutopic endometrium epithelial cells, normal endometrium epithelial cells co culture. Western blot (Western Blot) detection of E-cadherin, N-cadherin, CK7, vimentin, S100A4 in Ishikawa cells, adenopathy in the intima epithelial cells, normal endometrium epithelial cells expression. The invasion ability of Ishikawa cells and macrophages co cultured with epithelial mesenchymal transformation (EMT) was detected by cell invasion test. The cell morphology and intercellular connections of Ishikawa cells and macrophages co cultured with epithelial mesenchymal transformation (EMT) were observed by common optical microscopy and transmission electron microscopy (TEM). The changes of cell proliferation and apoptosis after epithelial mesenchymal transformation (EMT) in Ishikawa cells and macrophages were detected by flow cytometry (FCM). Finally, the changes of signal pathways in the process of epithelial mesenchymal transition (EMT) during co culture of Ishikawa cells and macrophages were detected by gene chip expression analysis. The results of the study: 1. Induction of epithelial mesenchymal transformation (EMT) in Ishikawa cells (EMT).2. macrophages induced adenomyosis in the endometrium and the epithelial cells of normal endometrium epithelial cell transformation (EMT) the potential of epithelial mesenchymal transition (EMT) in.3. endometrium is more significant than that of normal endometrium. Conclusion: macrophages can be induced. Epithelial mesenchymal transformation (EMT) in endometrium epithelial cells. Compared with normal endometrium, the eutopic endometrium in adenopathy is more potential for epithelial mesenchymal transition (EMT) after co culture with macrophages. Third part of the co culture system induces macrophage to M2 polarization: 1. to explore whether Ishikawa cells can induce giant cells. A study on whether macrophages can induce macrophage polarization in the endometrium and normal endometrium of adenomyosis to M2 polarization.2.. Methods: detection of the macrophages by flow cytometry (FCM) and polymerase chain reaction (PCR) with Ishikawa cells, adenomyosis in intima epithelial cells, and normal endometrium epithelial cells before and after co culture The expression of CD163, IL-10, MMP12. Results: 1.Ishikawa cells induced macrophages to M2 polarization.2. endometriosis in endometrium and normal endometrium can induce macrophage to M2 polarization.3. and adenomyosis eutopic endometrium, normal endometrial epithelial cells induce macrophage to M2 polarization more obvious. Conclusion: Ishikawa Cells, adenomyosis in the endometrium and normal endometrium epithelial cells can induce the transformation of macrophage to M2 type. At the same time, the effect of normal endometrial epithelial cells to induce macrophage polarization to M2 is stronger than that of endometrial epithelial cells in adenomyosis.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.71

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