2018-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/711013摘要：正常的滋養層細胞侵襲，是胚胎成功著床的重要因素。目前兩大妊娠期的殺手，子癲前症及植入性胎盤，一個是導因於滋養層細胞侵襲不足，另一個則是侵襲過度。在我們過去的計劃中，我們已經證實微小核糖核酸-7藉由負調控上皮細胞中胚轉化反應(EMT)，去調節滋養層細胞的侵襲能力。在本計劃中，我們則欲建立微小核糖核酸-7在正常妊娠的初孕、中孕及晚孕的血清表達量正常值，另外收集子癲前症及植入性胎盤的病人血清，偵測其微小核糖核酸-7變化，期望能運用微小核糖核酸-7，作為滋養層細胞侵襲異常疾病的生物標記物。本計劃的第一年，我們將收集30名正常孕婦、30名子癲前症、30名前置胎盤、30名植入性胎盤病人，經迴歸分析去建立微小核糖核酸-7變化的在三個孕期的表達量曲線，抽血時間分別在(1)初唐篩檢(2)妊娠糖尿病篩檢(3)生產前；建立微小核糖核酸-7於三個孕期分布的變化後，在第二年，我們進一步再去蒐集10名正常孕婦、10名子癲前症、10名前置胎盤、10名植入性胎盤病人，去驗證第一年所建立的微小核糖核酸-7分布值，確定其可當做滋養層細胞侵襲異常疾病的生物標記物。我們並在這些產婦生產時收集胎盤，使用螢光原位染色套件，去偵測微小核糖核酸-7的生體差異表達。本實驗中，微小核糖核酸-7的血清表達量偵測，主要使用偵測微小核糖核酸-7的商業套件(Qiagen)及生物條形碼凝膠分析模式，我們並將與生物科技公司(MicroRNA-Phalanx Biotech Company)研發一簡易型的微小核糖核酸-7偵測晶片。在確立微小核糖核酸-7與滋養層細胞侵襲異常疾病的相關性後，我們將針對胎盤中，微小核糖核酸-7下游標的物，如mTOR及 NF-B，進行免疫染色及西方墨點分析，期望找出微小核糖核酸-7調控滋養層細胞侵襲異常疾病的可能機轉。吾人亦使用滋養層細胞株，將aON-miR-7的轉導去誘導微小核糖核酸-7過度表達，或鎖定核酸技術(LNA)去向下調控微小核糖核酸-7，去看mTOR及NF-B表達變化，並觀察其與侵襲能力改變的相關。本兩年度的計劃，期望建立一個相對較低成本、高通量、或晶片為主的方式，去偵測病人血清中微小核糖核酸-7變化，以期輔助子癲前症及植入性胎盤的臨床診斷。並期未來發展預防或治療此滋養層細胞侵襲異常疾病的對策。<br> Abstract: Trophoblast invasion is crucial to establish placental circulation and ensure the nourishment for the developing embryos in mammals. Defect in trophoblast invasion may result into shallow implantation and subsequent fetal growth restriction and preeclampsia. In contrast, morbidity adherent placenta occurs if the trophoblast invasion is beyond normal depth in decidua. In our previous studies, we have discovered microRNA-7 as a novel player linking trophoblast invasion and negative regulation of epithelial-mesenchymal transition. In this project, we intend to establish a nomogram for the microRNA-7 in the first, second and third trimester. Besides, we will also enroll trophoblast invasion disorders such as preeclampsia and abnormally invasive placenta (AIP; placenta accreta) to determine their microRNA-7 transcription levels. Efforts are to use microRNA-7 as disease biomarkers for early detection of these diseases. In the first year, we will enroll 30 patients without pregnancy complications, 30 patients with abnormally invasive disorders (AIP; placenta accreta, increta, and percreta), another 30 patients with placenta previa without AIP, and another 30 patients with preeclampsia for establishment of nomogram of miR-7 expression in either physiological or pathological pregnancies. The time points of blood sampling are (1) 11-13 week when perform first trimester Down syndrome screening or routine prenatal blood test, (2) 24-28 week while performing 75g Glucola test ; (3) third trimester just before delivery. The placenta is also collected during delivery. The second phase (second year) will be the phase for biomarker validation. We will enroll 10 healthy pregnancies, 10 patients with placenta previa, another 10 patients of placenta previa with AIP; and 20 patients with preeclampsia from our department.Detection of miR-7 and the expression change of patient serum will be used via qPCR by commercial kits of miRNeasy Serum/Plasma Kit (Qiagen) and bio-barcode gel assay to determine the optimal method for future clinical applications. We also contacted the MicroRNA-Phalanx Biotech Company to develop a custom microarray chip for easy detection of miR-7 from serum sample. Simultaneously, we use the miRCURYLNA detection probe to detect miR-7 expression in placenta specimen from patients and normal control. After confirming the miR-7 expression changes contributing in the development of preeclampsia and AIP, we then use immunocytochemistry and western blotting to detect the differential expression of the downstream effector of miR-7 (such as mTOR and NF-B) either in placenta from patients or trophoblastic cell lines treated with overexpression or downregulation of microRNA-7. Through the effort of this two-year project, we wish to determine an easy-performed high-throughput, chip-based method to assist the diagnosis of preeclampsia and placenta accreta.滋養層細胞侵襲微小核糖核酸-7生物標記物子癲前症植入性胎盤基因晶片trophoblastinvasionmicroRNA-7biomarkerpreeclampsiaplacenta accretamicroarray