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    Title: 發炎對類上皮子宮內膜幹細胞之影響與子宮內膜異位症形成之相關性
    Authors: 何欣穎
    Contributors: 醫學科學研究所
    Date: 2010
    Issue Date: 2010-08-31 10:54:53 (UTC+8)
    Abstract: 子宮內膜異位症是指子宮內膜的組織生長至子宮內膜以外。此疾病影響了近10% 生育期的婦女。其症狀包括持續性腹痛、不正常的子宮出血與不孕。子宮內膜基層中的幹細胞被認為可以調控子宮內膜的再生並與子宮內膜異位症的發生有關。然而，其轉型成子宮內膜異位症的機制仍然是未知的。在臨床上發現慢性發炎與子宮內膜異位症是息息相關的。許多細胞激素，例如IL-1,6,8,10、TNF-α與VEGF在子宮內膜異位症的婦女腹腔液中是明顯增加的。基於以上論述，我們假設發炎可能誘導子宮內膜中的幹細胞轉型，進而促使子宮內膜異位症的發生。為了證明以上假設，我們收集臨床檢體包含 37位子宮內膜增生，43位巧克力囊腫與8位子宮肌腺症病患。藉由比對臨床檢體中的幹細胞多型性基因 (Oct-4與Nanog) 及其CA125分數發現，與子宮內膜增生的病患相比，巧克力囊腫與子宮內膜肌腺症病患有較高的Oct-4與Nanog表現，同時與其CA125有關。此外，我們建立一個無血清的老鼠類上皮子宮內膜幹細胞的培養環境。老鼠類上皮子宮內膜幹細胞強烈表現幹細胞特性的細胞表面標記Oct-4與SSEA-1，與幹細胞調控有關的細胞表面標記IGF-1/IGF-1R，以及表現弱幹細胞特性的CD49f、CD34、CD133、α-SMA與p63。利用即時聚合酶連鎖反應分析，在發炎刺激下，Oct-4、Sox2、c-Myc、Klf-4與IGF-1表現增加，顯示類上皮子宮內膜幹細胞的幹性有增加的趨勢。此外，在發炎反應下，老鼠類上皮子宮內膜幹細胞的E-cadherin表現下降伴隨N-cadherin表現上升，暗示EMT的發生。接著，為了觀察發炎對RL95-2細胞株 (低Oct-4表現的子宮內膜癌細胞株) 的影響，我們分別轉染Promotor-Oct-4-EGFP 質體與HA-Oct-4質體至RL95-2細胞株。在發炎情況下，Promotor-Oct-4-EGFP RL95-2細胞株觀察到較高的螢光表現，顯示較高的Oct-4表現。同時，在HA-Oct-4 RL95-2細胞株亦觀察到較高的HA與Oct-4蛋白表現。利用即時聚合酶連鎖反應分析，HA-Oct-4 RL95-2細胞株在發炎環境下具有較高的Oct-4與N-cadherin表現。這些結果暗示在發炎刺激下，幹細胞特性會增加與EMT現象的產生。總結，在我們的實驗中，我們證明慢性發炎主要透過誘導子宮內膜上皮細胞的轉型，對於子宮內膜異位症的發生與進展扮演著重要的角色。此結果對於未來不孕症與癌症發展提供了可能的治療標的。

    Endometriosis is the growth of endometrial tissues eutopically and/or ectopically. This disease affects nearly 10% of reproductive aged women with symptoms of persistent pelvic pain, abnormal uterine bleeding, and infertility. Stem cells in endometrial basalis layer have been hypothesized to mediate endometrium regeneration as well as the initiation of endometriosis. However, mechanism involved in endometriosis transformation processes still remains largely unknown. In clinic observation, chronic inflammation is tightly associated with endometriosis. Several cytokines including interleukin (IL)-1, 6, 8, 10, tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF) have been reported to be increased in the peritoneal fluid (PF) of women with endometriosis. In this aspect, we hypothesized that inflammation may result in transformation of endometrium stem cells and initiate the processes of endometriosis. To test this hypothesis, tissues with hyperplasia (n = 37), endometriosis (n = 43), and adenomyosis (n = 8) were collected and the clinical CA125 score was examined. Tissues were then analyzed to correlate with the pluripotent gene expression (Oct-4 and Nanog) with endometriosis progression in human. In our results, while comparison with the hyperplasia tissues, tissues with endometriosis/adenomyosis showed a higher expression of Oct-4 and Nanog as well as correlation with CA125. Furthermore, we established a serum-free culture system to generate mouse epithelial-like endometrium stem cells (mEESCs). These mEESCs showed a strong expression of stem cell markers (such as Oct-4 and SSEA-1) as well as IGF-1/IGF-1R (stemness regulatory signaling); and a weak expression of CD49f, CD34, CD133, α-SMA, and p63. Interestingly, by real-time PCR analysis, the mEESCs showed an increasing of stemness (such as Oct-4, Sox2, c-Myc, Klf-4, and IGF-1) under the treatment of inflammation condition medium (CM). Under the inflammation treatment, a down-regulation of E-cadherin transcription along with the up-regulation of N-cadherin transcription was observed, suggesting EMT processes of mEESCs. Furthermore, to investigate inflammation effect on the RL95-2 cell line (low Oct-4 expression endometrium carcinoma cell line), we transfected a Promotor-Oct-4-EGFP plasmid and a HA-Oct-4 plasmid individually into RL95-2 cell line. A higher GFP expression was observed under the inflammation treatment, indicating a stronger Oct-4 expression. Also, a higher Oct-4 and HA protein expression were detected by western blot in the HA-Oct-4 RL95-2 cell line under the inflammation condition. An increasing expression of Oct-4 and N-cadherin mRNA were showed in the HA-Oct-4 RL95-2 cell line. These results suggested increasing of stemness characteristic and EMT processes under treatment of the inflammation condition medium. In conclusion, we demonstrated that chronic inflammation processes might play an important role in initiation and development of endometriosis by transforming of endometrium epithelial stem cells. This observation may provide the possible therapeutic target of infertility and tumorigenesis.
    Relation: 56頁
    Description: 致謝 III
    摘要 IV
    ABSTRACT VI
    ABBREVIATIONS VIII
    CHAPTER 1 INTRODUCTION 1
    Human endometrium and menstrual cycle 1
    Stem cells 4
    Stem cells in human endometrium 5
    Markers of endometrium stem cells 6
    Stemness-related transcription factors in human endometrium 7
    Stem cells in mouse endometrium 8
    Endometrial stem cells and gynaecological disease 9
    Relationship between inflammation and endometriosis 10
    Relationship between inflammation and cancer 13
    Epithelial-mesenchymal transition 15
    Rationale and specific aims 16
    CHAPTER 2 MATERIALS AND METHODS 17
    Experimental animal 17
    Cultivation of mouse epithelial-like endometrial stem cells in serum-free culture medium 17
    Cultivation of mouse epithelial-like endometrial stem cells in serum-free culture medium under inflammatory conditioned medium 18
    Preparation for inflammatory conditioned medium 18
    RNA isolation and RT-PCR 18
    Polymerase Chain Reaction 19
    Real-time Polymerase Chain Reaction 19
    Immunocytochemical staining 20
    Human tissues 20
    Immunohistochemical staining 21
    Transfection 21
    HA-Oct-4 plasmid 22
    Western blotting 22
    CHAPTER 3 RESULTS 24
    Expression of pluripotent genes Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cysts) tissues in patients age of 25-35 by RT-PCR analysis 24
    Expression of pluripotent genes Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cysts) tissues in patients age of 35-40 and over 40 by RT-PCR analysis 24
    Expression of pluripotent transcription factor Oct-4 in endometriosis (chocolate cyst) tissue 25
    Cultivation of epithelial-like endometrial stem cells (mEESCs) in serum-free culture condition 26
    Identification of mouse epithelial-like endometrial stem cells (mEESCs) by immunochemical staining 26
    Effect of inflammation on mouse epithelial-like endometrial stem cells (mEESCs) 26
    Effect of inflammation on Promotor-Oct-4-EGFP RL95-2 cell line 27
    Effect of inflammation on HA-Oct-4 RL95-2 cell line 28
    CHAPTER 4 DISCUSSION 30
    CHAPTER 5 FIGURES 34
    Figure 1. Expression of Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cyst) tissues in patients age of 25-35. 34
    Figure 2. Expression of Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cyst) tissues in patients age of 36-40. 35
    Figure 3. Expression of Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cyst) tissues in patients age of over 40. 36
    Figure 4. Expression of pluripotent transcription factor Oct-4 in endometriosis (chocolate cyst) tissue (immunohistochemistry staining). 37
    Figure 5. Effect of coating materials on cultivation of mouse epithelial-like endometrial stem cells (mEESCs) in serum-free culture condition. 38
    Figure 6. Identification of transcription factors on mouse epithelial-like endometrial stem cells (mEESCs) by immunocytochemical staining. 39
    Figure 7. Effect of inflammation on mouse epithelial-like endometrial stem cells (mEESCs). 41
    Figure 8. Effect of inflammation on Promotor-Oct-4-EGFP RL95-2 cell line. 43
    Figure 9. Effect of inflammation on HA-Oct-4 RL95-2 cell line. 46
    SUPPLEMENTS 49
    Table 1. Expression of epithelial-like endometrial stem cells cell surface markers 49
    Table 2. PCR primer and product size 50
    Table 3. Real-time PCR primer 51
    REFERENCES 52


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