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姓名	賴宗炫(Tsung-Hsuan Lai)  查詢紙本館藏	畢業系所	系統生物與生物資訊研究所
論文名稱	探討人類子宮內膜 L-selectin ligands 在月經週期的表現 (Exploring the expression of L-selectin ligands in human endometrium throughout the menstrual cycle)
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摘要(中)	人類胚胎著床過程是透過胚胎和子宮內膜分泌產生的各種因子為媒介產生交互作用。成功的著床需要發育良好的胚胎和荷爾蒙調校的子宮內膜精細的同步化完美配合，然而關於胚胎著床的基本機制到目前為止仍未得到解決。胚胎著床涉及到胚胎滋養層細胞和子宮內膜上皮表面細胞黏著作用。證據顯示細胞黏著分子在胚胎著床過程中扮演重要角色。細胞黏著分子家族包含四個主要成員：Integrins，Cadherins, Selectins 和 Immunoglobulins。這些細胞黏著分子表面配體 (ligand) 是醣蛋白成分，成為細胞之間黏著的媒介。其基本功能包括維持組織完整性、傷口癒合、細胞型態運動、細胞移動及腫瘤轉移。最近研究人員已經注意到L-selectin ligand (LSL)在子宮內膜受孕性的角色。這幾年關於人類子宮內膜表現LSL的研究報告指出，LSL在子宮內膜受孕性及胚胎成功著床方面扮演重要角色。LSL可成為評估子宮內膜受孕性和胚胎著床的生物標記。 然而到目前為止，我們對於人類子宮內膜細胞分泌的LSL所知甚少。而且，對於參與合成人類子宮內膜細胞LSL的基因表現仍是一無所知。本研究嘗試找出人類子宮內膜細胞LSL的種類，探討這些LSL分子在正常人自然月經週期不同時期的表現型態。同時比較正常生育能力與患有子宮肌腺症婦女的子宮內膜LSL的表現差異。 我們收集41位具正常月經週期及生育力的子宮肌瘤病人、42位子宮肌腺瘤病人、和11位停經婦女的子宮內膜檢體。採用免疫染色、西方墨點法、和反轉錄聚合酶鏈式反應來評估LSL的表現。統計以無母數Kruskal-Wallis檢定法分析LSL在月經週期各分期的表現。 以MECA-79抗體做免疫染色分析顯示LSL在自然週期分泌早期至分泌中期有強度表現，在停經子宮內膜具低度表現。反轉錄聚合酶鏈式反應結果發現在生殖年齡期婦女、子宮肌腺症患者及停經婦女皆可發現5種LSL基因表現：PODXL， EMCN， CD300LG， GLYCAM1和CD34。EMCN在自然月經週期增殖期和分泌早期有顯著差異表現 (P<0.05)。EMCN在自然月經週期子宮內膜受孕姓可能扮演重要角色。針對子宮肌腺症檢體分析，免疫染色結果顯示LSL在月經週期各分期呈現低度表現。但從增值期至分泌晚期LSL表現上升，唯獨在分泌中期(胚胎著床期)LSL表現下降。LSL平均histological scores (HSCOREs)分數在增值期與分泌前期和分泌晚期具統計顯著差異(P<0.05)。5種LSL基因在各分期呈現差異性表現，其中PODXL在各分期之間具有顯著差異表現(P<0.05)。上述結果暗示子宮肌腺症可能影響分泌中期子宮內膜LSL的產生，進而使子宮內膜受孕性和胚胎著床受到不良影響。 未來需要更多生體和體外實驗研究LSL系統相關機轉，以決定其在子宮內膜受孕性所扮演的真正角色。
摘要(英)	In humans, embryo implantation is mediated by a variety of factors which are produced by both endometrium and blastocyst. Successful implantation requires a finely tuned synchrony between the embryo and receptive endometrium. However, fundamental questions about implantation remain unresolved. Initial step of embryo implantation is cell adhesion of trophectoderm of blastocyst and endometrial luminal epithelial cells of uterus, at their respective apical cell surfaces. Evidence suggests that the cell adhesion molecules play a unique role in human embryo implantation. The cell adhesion molecule family is composed of four members known as integrins, cadherins, selectins and immunoglobulins. These surface ligands, usually glycoproteins, mediate cell-to-cell adhesion. Their classical functions include maintenance of tissue integration, wound healing, morphogenic movements, cellular migrations and tumor metastasis. Recently, researchers have given attention to the role of L-selectin ligands (LSLs) in endometrial receptivity. The related works on the expression of LSLs in human endometrium in recent years have addressed the important role of LSLs in endometrial receptivity and successful implantation. LSLs may serve as a biomarker of endometrial receptivity and may help elucidate the implantation process. However, to date, little is known about the types of LSLs in human endometrium. Furthermore, the expressions of the genes involved in the synthesis of LSLs are yet available in human being. In this study, we intend to identify the LSL genes in human endometrium and to study the expression patterns of LSLs in the fertile patients compared with the patients with adenomyosis. We recruited 41 endometrial samples from reproductive-aged women with leiomyoma, 42 endometrial samples from patients with adenomyosis, and 11 endometrial samples from menopausal women. Immunohistochemistry, western blotting, and RT-PCR were performed to evaluate LSL expression. A non-parametric Kruskal-Wallis one-way analysis of variance with multiple comparisons was performed to examine differences among menstrual phases. Immunohistochemistry analysis with MECA-79 Ab revealed strong LSL expression from the early through the mid-secretory phase in natural cycle and low expression in menopausal endometrium. Five LSL genes were found in reproductive, adenomyotic and menopausal endometrium by RT-PCR: PODXL, EMCN, CD300LG, GLYCAM1, and CD34. EMCN differed significantly between the proliferative and early-secretory phases in natural cycle (P<0.05). The significant expression of EMCN between the proliferative and early-secretory phases might play a vital role in endometrial receptivity in natural cycle. In adenomyosis, Immunohistochemistry showed that LSL is expressed with weak intensity in the endometrium in all phases. In the luminal epithelium, LSL expression increased from the proliferative to the late-secretory phase but decreased in the mid-secretory phase (the period of implantation). There were significant differences in the mean histological scores (HSCOREs) among the proliferative, early-secretory, and late-secretory phases (P<0.05). The expression patterns of five LSL genes occurred differentially among phases. Moreover, PODXL differed significantly among phases (P<0.05). The results showed that adenomyosis may cause abnormalities in LSL production in the nid-secretory phase, which may contribute to impaired endometrial receptivity and implantation failure. Further studies in vitro and in vivo are required to determine the mechanisms related to the LSL system in human endometrium and to determine its role in endometrial receptivity.
關鍵字(中)	★ 子宮內膜 ★ 月經週期 ★ 子宮肌腺症 ★ 子宮肌瘤	關鍵字(英)	★ L-selectin ligand ★ endometrium ★ adenomyosis ★ leiomyoma ★ menstrual cycle
論文目次	中文摘要 I ABSTRACT III TABLE OF CONTENTS VI LIST OF TABLE IX LIST OF FIGURE X LIST OF SUPPLEMENTS TABLE XI LIST OF SUPPLEMENTS FIGURE XII CHAPTER 1. INTRODUCTION 1 CHAPTER 2. MATERIALS AND METHODS 6 2.1 SAMPLE COLLECTION 6 2.1.1 NORMAL ENDOMETRIAL SAMPLES COLLECTED FROM PATIENTS WITH LEIOMYOMA 6 2.1.2 ADENOMYOTIC ENDOMETRIA COLLECTED FROM PATIENTS WITH ADENOMYOSIS 7 2.1.3 ENDOMETRIAL SAMPLES WITHOUT SEX HORMONE STIMULATION COLLECTED FROM MENOPAUSAL WOMEN 7 2.2 IMMUNOHISTOCHEMISTRY 8 2.3 REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) 9 2.4 WESTERN BLOT ANALYSIS 11 2.5 STATISTICS 13 CHAPTER 3. RESULTS 14 3.1 STRONG LSL EXPRESSION WAS FOUND FROM THE EARLY TO MID-SECRETORY PHASE OF THE NATURAL CYCLE. FIVE LSL GENES WERE DETECTED IN HUMAN ENDOMETRIUM AND THE EXPRESSION PATTERNS REVEALED VARIATION AMONG THE PHASES. 14 3.1.1 DEMOGRAPHIC DATA OF THE SUBJECTS AT DIFFERENT PHASES OF THE NATURAL CYCLE 14 3.1.2 MEAN EPITHELIAL LUMINAL HSCORE HAS SIGNIFICANT DIFFERENCES AMONG PROLIFERATIVE, EARLY SECRETORY PHASES, MID-SECRETORY PHASES, AND LATE SECRETORY PHASES 15 3.1.3 FIVE LSL GENES WERE DETECTED IN HUMAN ENDOMETRIUM AND THE EXPRESSION PATTERNS REVEALED VARIATION AMONG THE PHASES. EMCN ARE SIGNIFICANTLY DIFFERENT BETWEEN THE PROLIFERATIVE AND EARLY-SECRETORY PHASES 17 3.2 LSL EXPRESSIONS WERE DOWNREGULATED IN THE LUMINAL EPITHELIUM OF ADENOMYOTIC ENDOMETRIA IN THE MID-SECRETORY PHASE. THE MRNA EXPRESSIONS OF THE LSL GENES OCCURRED DIFFERENTIALLY AMONG THE PHASES, BUT ONLY PODXL DIFFERED SIGNIFICANTLY. 22 3.2.1 DEMOGRAPHIC DATA OF THE SUBJECTS WITH ADENOMYOSIS AT DIFFERENT PHASES OF THE MENSTRUAL CYCLE 22 3.2.2 LSL WAS WEAKLY EXPRESSED IN THE LUMINAL AND GLANDULAR EPITHELIUM OF THE ENDOMETRIUM IN ALL PHASES AND LSL WERE DOWNREGULATED IN THE LUMINAL EPITHELIUM OF ADENOMYOTIC ENDOMETRIA IN THE MID-SECRETORY PHASE 24 3.2.3 FIVE LSL GENES WERE DETECTED IN ADENOMYOTIC ENDOMETRIA AND PODXL DIFFERED SIGNIFICANTLY AMONG THE PHASE 28 CHAPTER 4. DISCUSSION AND CONCLUSION 33 4.1 FIVE LSL GENES INCLUDING PODXL, EMCN, GLYCAM1, CD300LG, AND CD34 COULD EXPRESS IN HUMAN ENDOMETRIUM THROUGHOUT THE NATURAL MENSTRUAL CYCLE. EMCN EXPRESS SIGNIFICANTLY BETWEEN THE PROLIFERATIVE AND THE EARLY-SECRETORY PHASES MIGHT HIGHLIGHT ITS POTENTIAL IMPORTANCE IN ENDOMETRIAL RECEPTIVITY. 33 4.2 LSL EXPRESSIONS WERE DOWNREGULATED IN THE LUMINAL EPITHELIUM OF ADENOMYOTIC ENDOMETRIA IN THE MID-SECRETORY PHASE. PODXL DIFFERED SIGNIFICANTLY AMONG THE PHASES. THE RESULTS SHOWED THAT ADENOMYOSIS MAY CAUSE ABNORMALITIES IN LSL PRODUCTION, WHICH MAY CONTRIBUTE TO IMPAIRED ENDOMETRIAL RECEPTIVITY AND IMPLANTATION FAILURE. 39 4.3 CONCLUSION 44 REFERENCE 46 SUPPLEMENTS 52 APPENDIX 62
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指導教授	凌慶東(Qing-Dong Ling)	審核日期	2018-6-22
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