子宮內膜異位症是一種常見的，但複雜的婦科疾病，發病機制不明。它的特點是異位生长的子宮內膜組織。目前已提出多种發病機制的相关理論。现广泛接受經典的Sampson经血逆行的理論，，子宮內膜細胞出现在腹腔中。然而，对于子宮內膜異位症的發展中出现的細胞和分子事件却研究甚少。血管生成在早期子宮內膜異位病灶的生長和发展中起關鍵作用。在缺氧條件下，低灌注的異位子宮內膜組織促进血管形成因子被激活，以建立新的血管來提供氧和營養物質。血管生成的確切病理機制目前仍不清楚。我們推測，氧化應激是子宮內膜異位症的血管生成和發展的關鍵。目前的研究只是表明氧化應激可能促進子宮內膜細胞的生長和粘附，加剧子宮内膜异位症。但是，子宮內膜異位症的氧化應激具体機制仍不清楚。 / 在這項研究中，我們旨在了解氧化應激在子宮內膜異位症的早期血管生成和發展的重要作用。我们利用實驗性子宮內膜異位症小鼠模型用来研究子宮内膜异位症潛在的氧化應激機制。活体成像显示在子宮內膜异位植入后2 -6小時可检测到高活性氧簇（ROS）。同時，植入位处的也可见巨噬細胞浸潤和HIF-1α的表達。緊接著1天之內血管生成細胞因子表達上調，植入后一周后即有新血管形成。這表明巨噬細胞可能在子宮內膜異位症的早期發展中啟動氧化應激和促進血管生成。為了驗證在氧化應激，血管生成，子宮內膜異位癥的發展中巨噬細胞的作用，，採用抗F4/80抗體造成巨噬細胞功能缺失和12/15 LOX基因敲除小鼠模型下的氧化應激失調兩種模型進行研究。 通過干擾和破壞巨噬細胞介導的氧化應激，在子宮內膜植入處ROS產物生成顯著減少。血管生成因子受到抑制且血管發育不全。子宮內膜異位病灶均小於對照組。這表明巨噬細胞是氧化應激中重要介質。他們在促進子宮內膜異位症的早期血管生成和發展中起重要作用。由PX-478，HIF-1α抑製劑介導的治療也在子宮內膜異位症模型中進行更深入地研究。在血管生成途徑中抑制HIF-1α可減小病灶的大小和抑制新血管形成，但不影響巨噬細胞浸潤和子宮內膜植入物中的活性氧的生產。這表明巨噬細胞和ROS作用於子宮內膜異位症的血管形成機制的上游。總之，我們證明了巨噬細胞介導的氧化應激在子宮內膜異位症的早期血管生成和發展中起重要作用。 / Endometriosis is a common but complex gynecological disorder of unknown pathogenesis. It is characterized by ectopic growth of endometrial tissues Many theories have been proposed to the pathogenesis of endometriosis. The classical Sampson’s theory of retrograde menstruation is widely accepted to determine the presence of endometrial cells in the peritoneal cavity. However, little is known on the cellular and molecular events in the development of endometriosis. Angiogenesis plays a key step in the early growth and survival of the endometriotic lesions. Under hypoxic condition, pro-angiogenic factors are activated in poorly perfused ectopic endometrial tissues in order to establish new blood vessel to supply oxygen and nutrients. The precise pathological mechanisms of this angiogenesis pathway are still unclear. We hypothesized that oxidative stress is critical for the angiogenesis and development of endometriosis. Current studies only suggested oxidative stress may increase growth and adhesion of endometrial cells, promoting endometriosis. However, the underlying mechanism of oxidative stress in endometriosis remains unclear. / In this study, we aimed to understand the important role of oxidative stress in early angiogenesis and development of endometriosis. Experimental endometriosis mouse model was used to determine the underlying mechanism of oxidative stress. By in vivo imaging, high reactive oxygen species (ROS) production in the endometrial implants was detected from 2 -6 hours of transplantation. Concurrently, macrophage infiltration and HIF-1α expression in the implants were anticipated. Subsequently, angiogenic cytokines were upregulated within 1 day and new blood vessels were formed at least 1 week after transplantation. This suggested that macrophage may initiate the oxidative stress surge and angiogenesis pathway in early development of endometriosis. To confirm the role of macrophage in the oxidative stress, angiogenesis and development of endometriosis, macrophage depletion by anti-F4/80 antibody and oxidative stress disruption by 12/15 Lox transgenic knockout mice were employed in the experimental endometriosis models. By depleting macrophage and disrupting macrophage to mediate oxidative stress, the ROS production was significantly decreased in the endometrial implants. Angiogenesis factors were suppressed and blood vessels were under-developed. The endometriotic lesions were smaller than control. This showed macrophage is the key mediator of oxidative stress. They played an important role on promoting early angiogenesis and development of endometriosis. Potential therapeutic treatment by PX-478, a HIF-1α inhibitor, was further investigated in the experimental endometriosis model. Inhibition of HIF-1α in the angiogenic pathway decrease the lesion size and new vessel formed, but macrophage infiltration and ROS production in the endometrial implants was not affected. This suggested that macrophage and ROS are the upstream mechanism of the angiogenic pathway in endometriosis. In conclusion, we demonstrated that oxidative stress mediated by macrophages play an important role in the early angiogenesis and development of endometriosis. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Man, Chi Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 66-82). / Abstracts also in Chinese. / Title --- p.i / Acknowledgment --- p.ii / List of publication and conferences --- p.iv / Additional achievements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xviii / Abstract --- p.xix / Chinese Abstract --- p.xxi / Chapter Chapter 1. --- Endometriosis / Chapter 1.1. --- Definition of Endometriosis --- p.1 / Chapter 1.1.1. --- Treatment of Endometriosis --- p.2 / Chapter 1.1.1.1. --- Expectant Management --- p.2 / Chapter 1.1.1.2. --- Medical Therapy --- p.2 / Chapter 1.1.1.2.1. --- Analgesics --- p.2 / Chapter 1.1.1.2.2. --- Hormones --- p.3 / Chapter 1.1.1.3. --- Surgery --- p.4 / Chapter 1.1.1.3.1. --- Surgical Treatment --- p.4 / Chapter 1.1.1.3.2. --- Conservative Surgery --- p.4 / Chapter 1.1.1.3.3. --- Definitive Surgery --- p.5 / Chapter 1.1.2. --- Pathogenesis and Pathophysiology of Endometriosis --- p.5 / Chapter 1.2. --- Oxidative stress --- p.7 / Chapter 1.2.1. --- Pro-oxidants --- p.7 / Chapter 1.2.2. --- Antioxidants --- p.8 / Chapter 1.2.2.1. --- Non-enzymatic Antioxidants --- p.9 / Chapter 1.2.2.2. --- Enzymatic Antioxidants --- p.9 / Chapter 1.2.2.2.1. --- Superoxide Dismutase --- p.10 / Chapter 1.2.2.2.2. --- Catalase --- p.10 / Chapter 1.2.2.2.3. --- Peroxiredoxins --- p.11 / Chapter 1.2.2.2.4. --- Glutathione Peroxidases --- p.11 / Chapter 1.2.3. --- Oxidative Stress and Diseases --- p.11 / Chapter 1.2.4. --- Endometriosis and Oxidative Stress --- p.12 / Chapter 1.2.5. --- Inflammatory Response --- p.13 / Chapter 1.2.6. --- Animal Models of Endometriosis --- p.14 / Chapter 1.3. --- Angiogenesis --- p.15 / Chapter 1.3.1. --- Angiogenesis and Diseases --- p.16 / Chapter 1.3.2. --- Animal Models of Angiogenesis --- p.17 / Chapter 1.3.3. --- Endometriosis and Angiogenesis --- p.18 / Chapter 1.3.4. --- Anti-angiogenesis Therapy --- p.19 / Chapter 1.3.5. --- Rodent Models of Endometriosis --- p.21 / Chapter Chapter 2. --- Objective and Hypothesis --- p.23 / Chapter Chapter 3. --- Methodology / Chapter 3.1. --- Mouse Models of Endometriosis --- p.24 / Chapter 3.1.1. --- Animals --- p.24 / Chapter 3.1.2. --- Ovariectomy --- p.24 / Chapter 3.1.3. --- Transplantation of Endometrium --- p.25 / Chapter 3.1.3.1. --- Donor mice --- p.25 / Chapter 3.1.3.2. --- Recipient mice --- p.25 / Chapter 3.1.4. --- Luciferase ⁺/⁺ transgenic mice --- p.26 / Chapter 3.1.5. --- Macrophage Depletion Model --- p.26 / Chapter 3.1.6. --- 12/15 Lox Transgenic Knockout Mice --- p.26 / Chapter 3.1.6.1. --- 12/15 Lox Knockout to Wildtype --- p.27 / Chapter 3.1.6.2. --- Wildtype to 12/15 Lox Knockout --- p.27 / Chapter 3.1.7. --- HIF-1α Inhibition Model --- p.27 / Chapter 3.2. --- Growth and Development of Endometriotic Lesions --- p.28 / Chapter 3.2.1. --- Termination --- p.28 / Chapter 3.2.2. --- Lesion Size --- p.28 / Chapter 3.2.3. --- In Vivo Non-Invasive Imaging --- p.28 / Chapter 3.2.3.1. --- Luciferase Imaging --- p.28 / Chapter 3.2.3.2. --- Oxidative Stress Imaging --- p.29 / Chapter 3.2.3.3. --- Angiogenesis Imaging --- p.30 / Chapter 3.2.4. --- Histological Evaluation --- p.30 / Chapter 3.2.5. --- Immunostaining --- p.30 / Chapter 3.2.5.1. --- Immunohistochemistry on Oxidative Stress Markers --- p.30 / Chapter 3.2.5.2. --- Immunohistochemistry on Macrophages and Neutrophils --- p.31 / Chapter 3.2.5.2. --- Immunofluorescence on New Vessel Formation --- p.32 / Chapter 3.2.6. --- Terminal Deoxynucleotidyltransferase (TdT)-mediated dUTP End Labeling (TUNEL) Assay --- p.33 / Chapter 3.2.7. --- Quantitative real-time --- p.33 / Chapter 3.2.7.1. --- qPCR on Apoptotic Factors --- p.33 / Chapter 3.2.7.2. --- qPCR on Hypoxic Factors --- p.34 / Chapter 3.2.7.3. --- qPCR on Angiogenic Factors --- p.34 / Chapter 3.2.8. --- 8-Isoprostane --- p.34 / Chapter 3.3. --- Reproductive safety of PX-478 --- p.35 / Chapter 3.4. --- Data Analysis --- p.36 / Chapter Chapter 4. --- Results --- p.37 / Chapter 4.1. --- Endometriosis Growth and Development --- p.37 / Chapter 4.1.1. --- In vivo Non-Invasive Imaging --- p.37 / Chapter 4.1.2. --- Ectopic Endometriotic Lesions --- p.37 / Chapter 4.1.3. --- Histological Examination --- p.37 / Chapter 4.1.4. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.38 / Chapter 4.1.5. --- Real-time PCR --- p.38 / Chapter 4.1.6. --- Oxidative Stress --- p.38 / Chapter 4.1.6.1. --- Non-invasive Imaging (IVIS) --- p.38 / Chapter 4.1.6.2. --- Measurement of 8-isoprostane --- p.39 / Chapter 4.1.6.3. --- Real-time PCR Analysis on Hypoxic Markers --- p.39 / Chapter 4.1.6.4. --- Immunohistochemistry (HIF-1α and VEGF) --- p.40 / Chapter 4.1.7. --- Angiogenesis --- p.40 / Chapter 4.1.7.1. --- Cellvizio Imaging --- p.40 / Chapter 4.1.7.2. --- Real-time PCR Analysis on Hypoxia & Angiogenic Markers --- p.40 / Chapter 4.1.7.3. --- Immunofluorescence --- p.41 / Chapter 4.2. --- Effects of Estrogen on Oxidative Stress and Angiogenesis --- p.41 / Chapter 4.3. --- Macrophage Depletion --- p.43 / Chapter 4.3.1. --- Lesion Growth and Development --- p.43 / Chapter 4.3.1.1. --- In Vivo Non-Invasive Imaging --- p.43 / Chapter 4.3.1.2. --- Ectopic Endometrium Lesions --- p.44 / Chapter 4.3.1.3. --- Histological Examination --- p.44 / Chapter 4.3.1.4. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.44 / Chapter 4.3.1.5. --- Real-time PCR --- p.45 / Chapter 4.3.2. --- Oxidative stress --- p.45 / Chapter 4.3.2.1. --- In Vivo Imaging --- p.45 / Chapter 4.3.2.2. --- 8-isoprostane --- p.45 / Chapter 4.3.2.3. --- Real-time PCR Analysis on Hypoxic Markers --- p.45 / Chapter 4.3.2.4. --- Immunohistochemistry --- p.46 / Chapter 4.3.3. --- Angiogenesis --- p.46 / Chapter 4.3.3.1. --- Real-time PCR analysis on angiogenic markers --- p.46 / Chapter 4.3.3.2. --- Immunofluorescence --- p.47 / Chapter 4.4. --- 12/15 Lox Transgenic Knockout --- p.47 / Chapter 4.4.1. --- Lesion Growth and Development --- p.47 / Chapter 4.4.1.1. --- Ectopic Endometrium Lesions --- p.47 / Chapter 4.4.1.2. --- Histological Examination --- p.48 / Chapter 4.4.1.3. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.48 / Chapter 4.4.1.4. --- Real-time PCR --- p.49 / Chapter 4.4.2. --- Oxidative Stress --- p.50 / Chapter 4.4.2.1. --- 8-isoprostane --- p.50 / Chapter 4.4.2.2. --- Real-time PCR Analysis on Hypoxic Markers --- p.50 / Chapter 4.4.2.3. --- Immunohistochemistry --- p.51 / Chapter 4.4.3. --- Angiogenesis --- p.51 / Chapter 4.4.3.1. --- Real-time PCR Analysis on Angiogenic Markers --- p.51 / Chapter 4.4.3.2. --- Immunofluorescence --- p.53 / Chapter 4.5. --- HIF-1α inhibition --- p.53 / Chapter 4.5.1. --- Lesion Growth and Development --- p.53 / Chapter 4.5.1.1. --- In Vivo Imaging --- p.53 / Chapter 4.5.1.2. --- Ectopic Endometrium Lesions --- p.54 / Chapter 4.5.1.3. --- Histological examination --- p.54 / Chapter 4.5.1.4. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.54 / Chapter 4.5.1.5. --- Real-time PCR --- p.55 / Chapter 4.5.2. --- Oxidative stress --- p.55 / Chapter 4.5.2.1. --- In Vivo Imaging --- p.55 / Chapter 4.5.2.2. --- 8-isoprostane --- p.55 / Chapter 4.5.2.3. --- Real-time PCR Analysis on Hypoxic Markers --- p.55 / Chapter 4.5.2.4. --- Immunohistochemistry --- p.56 / Chapter 4.5.3. --- Angiogenesis --- p.56 / Chapter 4.5.3.1. --- Real-time PCR Analysis on Angiogenic Markers --- p.56 / Chapter 4.5.3.2. --- Immunofluorescence --- p.57 / Chapter 4.5.4. --- Safety Assessment of PX-478 --- p.58 / Chapter Chapter 5. --- Discussion --- p.59 / Chapter 5.1. --- Animal Model and Transplantation Method --- p.59 / Chapter 5.2. --- Oxidative Stress and Its Role in Early Development of Endometriosis --- p.59 / Chapter 5.3. --- Macrophage and Oxidative Stress in Endometriosis --- p.60 / Chapter 5.4. --- Potential Antioxidative Therapy for Endometriosis Treatment --- p.61 / Chapter 5.5. --- Safety --- p.62 / Chapter 5.6. --- Limitation and Further Studies --- p.63 / Chapter Chapter 6. --- Conclusions --- p.65 / Chapter Chapter 7. --- References --- p.66
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328555 |
| Date | January 2013 |
| Contributors | Man, Chi Wai., Chinese University of Hong Kong Graduate School. Division of Obstetrics and Gynaecology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xxii, 169 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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