| 摘要: | 子宮內膜異位症在臨床及體外試驗指出子宮內膜異位細胞 (endomeriotic cells)具有入侵及轉移生長能力，但目前致病的因子與分子機轉仍未清楚。先前本實驗室已證實患有子宮內膜異位症的病患當中，骨橋蛋白 (osteopontin，OPN) 會大量表達於子宮異位組織及腹膜液中，並且與integrin αvβ3結合促進內膜異位細胞移行。OPN被證實可與細胞表面接受器integrin或CD44交互作用，進而調節細胞貼附、移行、分化、存活等功能。OPN 被發現具有三種異構體 (isoform)，分別為OPNa (full length)、OPNb、OPNc。OPNc isoform 相較於全長OPNa 對乳癌細胞轉移的影響更為顯著。在腫瘤幹細胞 (tumor stem cell) 富含帶有CD44+ 的細胞族群，CD44 的表達與癌細胞轉化為間質細胞 (mesenchymal cell) 進而促進細胞移行有關。此外，高表現的 CD44量常伴隨著惡性腫瘤的低治癒率。因此，本論文主要探討OPN是否會透過與CD44的相互作用，而促進子宮內膜異位細胞發生上皮細胞間質轉化 (epithelium mesenchymal transition, EMT)，增進細胞移行並於異位處生長。首先，利用Q-PCR及西方墨點法發現子宮肌腺症 (Adenomyosis) 和巧克力囊腫 (Chocolate cyst) 病患的組織中OPN 、CD44 及EMT標記蛋白的大量表達。此外， OPN異構體mRNA會特異性於子宮肌腺症檢體中大量表達。我們進而製備並轉殖 OPNb 及 OPNc 於子宮內膜異位細胞，觀察到隨著時間增長OPN及OPNc 影響細胞型態改變。近一步分析型態改變的細胞中是否有 EMT 標記分子的表達，結果顯示高表現OPN異構體的細胞CD44v異構體及 EMT 標記蛋白N-cadherin、ICAM 和 VCAM 的表達都有增加的現象，反之 E-cadherin 表現減少。為了釐清 OPN 是否透過 CD44 或 integrin αvβ3 的交互作用進而活化PI3K or NF-κB 訊息路徑進而產生影響，我們使用knockdown CD44、αvβ3抗體或以the PI3K or NFκB抑制劑處理後發現，受抑制的 CD44或integrin αvβ3及抑制劑的使用會影響 OPN 異構體對細胞 EMT的表現及移能力的影響。未來期望能應用阻斷OPN 與 CD44 或 integrin αvβ3 的交互作用於子宮內膜異位症的臨床治療。
Endometriosis is a complex gynecologic disorder. Susceptibility to endometriosis depends on the complex interaction of genetic, immunologic, hormonal, and environment factors but the process and the molecular mechanism of pathogens is remains unclear. In our previous finding, osteopontin (OPN) was enhanced in the endometriotic tissues and contributes to cell migration and invasion via interacting with integrin αvβ3. Three types of OPN isoforms, OPNa (OPN full length) OPNb, OPNc were identified. Research found that OPNc isoform was more impetus in cancer metastasis than OPNa. The migratory effects of osteopontin were identified to be mediated through integrin αvβ3 and CD44 receptors in several cancer progression. Recent evidence suggests that a subpopulation of CD44+ cells attribute of cells that undergo an epithelial-mesenchymal transition (EMT), leading to tumour metastasis, and resulting a worse prognosis. In this study, we plan to investigate whether OPN interacts with CD44 contributing to endometriotic migration and EMT. In this study, variable OPN isoforms, CD44, and EMT markers were identified in endometriotic tissues especially significant increase in adenomyotic lesion. Furthermore, the vectors containing the human OPN splice variants (OPNb or OPNc) were transfected into the endometriotic cells. OPN stimulated CD44 expression and significantly induced morphology changes. Hence, the CD44 SiRNA, αvβ3 antibody, Wortmanin and CAPE were used to clarify wheher OPN interacts with CD44 receptor or integrin αvβ3 through activation the PI3K or NF-κB molecular signaling pathways lead to regulating cell migration, actin remodeling and EMT. The results will unravel the causal role of OPN in the regulation of endometriotic cell growth, which may promote the development of may promote the development of OPN directed therapies for patients with endometriosis. |
