發炎細胞激素與多功能性轉錄因子OCT4對子宮內膜異位症形成之探討

Role of Inflammatory Cytokines and Pluripotent Factor OCT4 in Endometriosis

李威進(Wei-Chin Lee)

指導教授 ： 黃彥華

臺北醫學大學/醫學院/醫學科學研究所/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00078

摘要

子宮內膜異位症是指子宮內膜組織生長在子宮腔以外之地方。而子宮內膜異位症患者其腹水 (peritoneal fluid) 中許多發炎細胞激素 (inflammatory cytokine) 有增加之情形。且多功能性轉錄因子 (pluripotent transcription factor) OCT4與子宮內膜異位症息息相關，但是目前OCT4基因在子宮內膜異位症中扮演的角色仍不清楚。在本論文中，我們探討發炎細胞激素和OCT4基因在子宮內膜細胞遷移的影響與內膜異位的相關性。為了驗證以上問題，我們收集94位臨床病人檢體，包含子宮內膜過度增生36位、子宮肌腺症30位、巧克力囊腫28位。透過即時定量聚合酶鏈鎖反應 (Q-PCR) 分析臨床檢體，發現子宮內膜異位症 (子宮肌腺症、巧克力囊腫) 的病人與子宮內膜過度增生之病人相比具有較高的OCT4、NANOG基因表現。免疫組織染色實驗證實子宮內膜異位症病人檢體組織切片中發現較高表現的OCT4蛋白。同時子宮內膜異位症病人檢體中發現OCT4基因表現與遷移基因VIMENTIN、TWIST、SNAIL、SLUG具有正相關性。在人類子宮內膜癌細胞株RL95-2與HEC1A中，添加發炎細胞激素後，透過即時定量聚合酶鏈鎖反應及OCT4啟動子報導冷光酵素活性測定 (OCT4 promoter reporter luciferase assay) 發現OCT4基因表現量有增加趨勢。接著我們發現高度表現OCT4蛋白的人類子宮內膜細胞珠 (RL95-2、HEC1A)，其遷移相關的基因VIMENTIN、TWIST、SNAIL、SLUG、N-CADHERIN與蛋白VIMENTIN、TWIST、SLUG、N-CADHERIN表現量皆有增加的趨勢。細胞劃痕癒合分析 (wound healing assay) 及細胞透孔遷移分析 (transwell migration assay) 進一步證實高度表現OCT4蛋白可以促使子宮內膜細胞表現較強的遷移能力，同時透過細胞染色和共軛焦顯微鏡發現高度表現OCT4蛋白會影響子宮內膜細胞內細胞骨架actin的分布且促使子宮內膜細胞外型改變。總結：我們發現多功能性轉錄因子OCT4在子宮內膜異位組織中具有較高的表現量，且促使子宮內膜細胞具有較強的遷移能力。由本篇論文研究可解釋部分子宮內膜異位形成機制。

關鍵字

子宮內膜異位症 ； 發炎 ； 發炎細胞激素 ； 多功能性轉錄因子OCT4 ； 遷移

並列摘要

Endometriosis is the ectopic growth of endometrial tissues. Some of inflammatory cytokines were elevated in endometriotic peritoneal fluid. The pluripotent transcription factor, OCT4, is associated with endometriosis. However, the role of inflammatory cytokines and OCT4 in endometriosis still remains largely unknown. In this study, we examined the roles of OCT4 in promoting the endometrial cell migration and the association of human ectopic endometriosis. In this study, we analyzed gene and protein expression levels of OCT4 and NANOG in patient tissues (n = 94) by a quantitative real-time Q-PCR and immunohistochemical staining. The OCT4, NANOG expressions were significantly increased in the ectopic endometriotic tissues (n = 58, 30 for adenomyosis tissues and 28 for chocolate cysts), comparing with the hyperplastic tissues (n = 36). The OCT4 protein was detected in both stromal and luminal epithelial cells in adenomyosis tissues and chocolate cysts. The OCT4 transcriptional level in endometriotic tissues was positively correlated with expressions of genes associated with cell migration, such as VIMENTIN, TWIST, SNAIL, and SLUG. We demonstrated that inflammatory cytokines increased the OCT4 expression in human endometrium RL95-2 and HEC1A cells using quantitative real-time Q-PCR and OCT4 promoter reporter luciferase assay. Overexpression of OCT4 in the human RL95-2 and HEC1A endometrial carcinoma cell lines (both of which have low OCT4 expression levels) significantly increased expression levels of migration-associated genes (VIMENTIN, TWIST, SNAIL, SLUG, and N-CADHERIN) and proteins (VIMENTIN, TWIST, SLUG, and N-CADHERIN), and promoted the migration of endometrial cells as evidenced by wound-closure and transwell assays. Immunocytochemical staining combined with confocal imaging further demonstrated that OCT4 affected the actin-filament distribution and changed the cell morphology of human endometrial cells into a migratory shape. In summary, the pluripotent transcription factor, OCT4, is significantly upregulated in ectopic endometriotic tissues and promotes the migratory ability of human endometrial cells. Findings in this study explain part of the molecular mechanism of the progression of human ectopic endometriosis.

並列關鍵字

endometriosis ； inflammation ； inflammatory cytokines ； OCT4 ； migration

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