缺氧在子宫内膜异位症中的致病作用及机制研究

发布时间：2018-04-26 18:45

本文选题：子宫内膜异位症 + 缺氧　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：背景子宫内膜异位症（endometriosis，EMs）是一种现代妇科病，在育龄期女性的发病率为6-10%，在不孕不育女性中可达30%。EMs患者中卵巢子宫内膜样癌和透明细胞癌发病风险明显增高。目前，腹腔镜手术与性激素的应用已成为EMs主要治疗方法，辅助生殖技术为患者改善生育状况带来希望，然而术后复发、粘连引起的盆腔疼痛及不孕，甚至远处转移及癌变严重损害着女性身心健康。 1927年Sampson提出的经血逆流种植学说至今仍被众多实验证据所支持，但经血逆流是妇女月经期常见的现象，而EMs发生率与其比较相对较低。近年来EMs“在位内膜决定论”的发病学说补充和发展了经血逆流理论。但是在位子宫内膜究竟通过什么途径“决定”了EMs何时发生及如何发展，尚无相关报道。EMs直接与月经相关，月经期间子宫螺旋动脉收缩，子宫内膜处于缺血、缺氧状态，内膜碎片随经血逆流至盆腔，在异位粘附、侵袭、血管形成之前均处于缺氧状态。近年研究发现缺氧影响着肿瘤的发生、发展；而EMs可以浸润、转移及复发，有类似恶性肿瘤的特性和生物学行为。因此，探索缺氧在EMs发生发展中的可能作用机制，对于解释EMs诸多生物学行为根源，寻找新的诊断、治疗和预防措施具有一定的理论和实践意义。目的研究持续缺氧以及急性缺氧对EMs细胞及组织增生，凋亡，血管生成等影响；探讨不同来源的子宫在位、异位内膜组织，，以及在SCID小鼠模型的不同移植部位，EMs病灶形成与缺氧、血管生成和粘连等相关因子的关系。方法原代培养人子宫内膜细胞，比较缺氧或者常氧培养对于不同来源子宫内膜细胞增殖凋亡的影响；通过SCID小鼠异种移植人子宫内膜建立EMs在体模型，观察经缺氧或高氧预处理移植物后病灶的生长情况；在SCID小鼠EMs模型观察EMs在位或异位内膜移植，以及SCID小鼠腹腔内或皮下不同部位EMs移植物的生长情况。应用细胞荧光免疫方法、流式细胞仪分析，HE染色，免疫组化技术，Western blot和ELISA等方法检测与增殖，凋亡，血管生成和缺氧等相关蛋白因子的表达。结果1、子宫内膜细胞在缺氧培养组比常氧培养组凋亡率上升；在缺氧环境下，EMs细胞伪足比对照组细胞形成增多，而凋亡率减少。缺氧培养使EMs细胞表达HIF-1α，VEGF，Ki67比常氧对照组增加。2、在SCID小鼠EMs模型中，经缺氧预处理后的移植物与高氧及常氧预处理对照组比较，移植物重量增加；缺氧预处理组病灶中的MVD、HIF-1α、VEGF和Ki67等蛋白表达增加。3、不同来源的内膜组织在SCID小鼠腹腔内生长方式不同，EMs异位内膜移植物的重量，组织中CD34、VEGF、HIF-1α和CA125表达以及SCID小鼠腹水中CA125含量比在位内膜组以及对照组内膜移植物明显增加。4、SCID小鼠腹腔内移植组织与网膜及腹膜形成粘连带，粘连带内可见血管生成；而皮下移植的组织粘连带形成相对较少。皮下移植病灶的体积增加明显，以及MMP-2和TIMP-2，MVD，VEGF和HIF-1α表达均高于腹腔内移植病灶。结论1、缺氧培养促使EMs内膜细胞凋亡的同时，也促使EMs内膜细胞表达HIF-1α、血管生成和增生相关蛋白；EMs细胞抗凋亡以及增生能力要高于对照内膜细胞。2、急性缺氧促进EMs移植病灶表达HIF-1α、血管生成以及增生相关因子，影响病灶生长。3、EMs异位内膜移植病灶生长能力要强于EMs在位内膜以及非EMs对照组内膜， EMs在位内膜与对照内膜也存在差别，异位内膜细胞可能是EMs在位内膜缺氧选择的结果。4、SCID小鼠不同部位的EMs移植物生长以及粘连带形成存在差异，粘连、血管生成以及HIF-1α等因子表达也不同，这说明局部缺氧微环境影响EMs病灶生物学行为。
[Abstract]:Background endometriosis (EMs) is a modern gynecologic disease. The incidence of women in the childbearing age is 6-10%. The risk of ovarian endometrioid and transparent cell carcinoma in the patients with infertile women is significantly higher than that of 30%.EMs patients. At present, the application of laparoscopic hand and sex hormone has become the main treatment of EMs. Assisted reproductive technology brings hope for the patients to improve their fertility. However, postoperative recurrence, pelvic pain and infertility caused by adhesion, even distant metastasis and canceration seriously damage the physical and mental health of women.
The theory of reflux planting for blood flow, proposed by Sampson in 1927, is still supported by numerous experimental evidence, but the reverse flow of blood is a common phenomenon in women's menstrual period, and the incidence of EMs is relatively low. In recent years, the theory of EMs "eutopic endometrial determinism" has been supplemented and developed by the theory of reverse flow of blood, but the eutopic endometrium is in fact By what means "determines" when and how EMs develops, there is no related reports that.EMs is directly related to menstruation, the uterine spiral artery contraction, the endometrium in the ischemic and anoxic state, the endometrium fragments with the menstrual flow to the pelvic cavity, in the anoxic state before ectopic adhesion, invasion and angiogenesis. Anoxia affects the occurrence and development of tumor, and EMs can infiltrate, metastases and relapse with the characteristics and biological behavior of malignant tumor. Therefore, it is possible to explore the possible mechanism of hypoxia in the development of EMs, and to explain the root of the biological behavior of EMs and to find new diagnosis, treatment and prevention measures. Practice meaning.
Objective to study the effects of continuous hypoxia and acute hypoxia on the proliferation, apoptosis and angiogenesis of EMs cells and tissues, and to explore the relationship between different sources of uterus, ectopic endometrium, different sites of transplantation of SCID mice, the formation of EMs focus and the related factors of hypoxia, angiogenesis and adhesion.
Methods the primary endometrium cells were cultured, and the effects of hypoxia or aerobia on the proliferation and apoptosis of endometrium cells in different sources were compared. EMs in the endometrium of the xenotransplantation of SCID mice was established to observe the growth of the lesion after the hypoxia or hyperoxia preconditioning of the graft, and the EMs in the EMs model of SCID mice was observed. The growth of EMs grafts in the abdominal or subcutaneous parts of SCID mice and the growth of transplanting or ectopic endometrium, and using cell fluorescence immunoassay, flow cytometry, HE staining, immunohistochemistry, Western blot and ELISA, were used to detect the expression of protein factors, such as proliferation, apoptosis, angiogenesis and hypoxia.
Results 1, the apoptosis rate of the endometrium cells in the hypoxia culture group was higher than that in the normal oxygen culture group. In the hypoxia environment, the cell formation of EMs cell hypoplastic cells increased and the apoptosis rate decreased. The hypoxia culture made EMs cells express HIF-1 alpha, VEGF, Ki67 more.2 than the normal oxygen control group. In the EMs model of SCID mice, the anoxic pretreated graft was found in the EMs model of SCID mice. Compared with hyperoxia and hyperoxia preconditioning group, the weight of graft increased, and the expression of MVD, HIF-1 a, VEGF and Ki67 increased.3 in the focus of the hypoxia preconditioning group. The growth patterns of endometrium in the abdominal cavity of SCID mice were different in different sources, the weight of EMs endometrium graft, CD34, VEGF, HIF-1 alpha and CA125 expression in the tissue and the small SCID. The content of CA125 in rat ascites was significantly increased by.4 than in the eutopic endometrium and the control group. The intraperitoneal transplantation tissue in SCID mice was visially connected with the omentum and peritoneum, and the vascular formation was visible in the adhesion zone; and the tissue adhesion zone formed by subcutaneous transplantation was relatively small. The volume of subcutaneous transplantation lesions increased significantly, and MMP-2 and TIMP-2, MV The expressions of D, VEGF and HIF-1 alpha were all higher than those in abdominal cavity.
Conclusion 1, hypoxia culture promotes the apoptosis of EMs intima cells, and also promotes the expression of HIF-1 alpha, angiogenesis and proliferation related proteins in EMs endometrium cells. The anti apoptosis and proliferation ability of EMs cells is higher than that of the control endometrial cells.2. Acute hypoxia promotes the expression of HIF-1 a in EMs transplant lesions, angiogenesis and proliferation related factors, which affect the growth of the lesion. The growth ability of.3, EMs ectopic endometrium transplantation focus is stronger than that of EMs eutopic endometrium and non EMs control group. EMs eutopic endometrium and control endometrium are also different. Ectopic endometrium cells may be the result of EMs eutopic endometrial anoxia selection,.4, EMs graft growth and adhesion formation in different parts of SCID mice, adhesions, and blood vessels The expression of HIF-1 and alpha is also different. This indicates that the local hypoxia microenvironment affects the biological behavior of EMs lesions.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.71

【参考文献】