2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656214摘要：滋養層細胞的侵襲對胚胎及胎盤的發育是一個最重要的步驟，細胞侵襲過淺可能導致子癲前症，而過度侵襲則導致植入性胎盤。我們已於過去的計劃中證實，滋養層細胞的侵襲與其自身的第一型轉化生長因子受體(TRI)的自體調控有相關，但確切的基因訊息傳遞與調控尚未明瞭。本計劃主要應用新興的微小RNA 的技術，去瞭解其在第一型轉化生長因子受體調控滋養層細胞的侵襲，所扮演的調節角色，並希望借助微小RNA 陣列的技術，找出最重要相關的微小RNA，期望可找到子癲前症的有效的微小RNA 生體標誌。在第一年的計劃中，我們使用滋養層細胞的細胞株與來自初期懷孕接受子宮擴括術的檢體，再以TRI 的特定RNAi 轉殖入慢病毒的載體，在細胞內大量表現去抑制TRI 在細胞內的表現，以達到增進滋養層細胞侵襲的效果。在以定量PCR 及西方墨點技術確認TRI 的表現確實減少後，將處理前後的檢體抽取RNA，送微小RNA 陣列分析，找出有顯著向上或向下調控的微小RNA。在第二年計劃中，我們將找到的可能相關的微小RNA，以質體載入轉殖到滋養層細胞中令其大量表達，或以微小RNA 海綿(sponge)的技術去抑制這些特定微小RNA 的表現，經由侵襲小室或實時顯微錄影去確認此特定微小RNA 確可調控滋養層細胞的侵襲。在最後一年計劃中，我們將抽取健康孕婦及子癲前症病人的血，去抽取RNA，再做吾人在前兩年鑑定出有相關的微小RNA 的實時PCR，去看何者的變化最為顯著，期望可做未來子癲前症病人的生體標誌，以利未來可提早偵測、甚至發展新藥預防。<br> Abstract: The invasion of trophoblast is a key step for the development of embryo andplacenta. Shallow invasion of trophoblast may cause preeclampsia, while deepinvasion results in placenta accreta of pregnancy. In our last project, we havedemonstrated that the trophoblast invasion is regulated by the TRI expression.However, how TRI expression exerts their signals to regulate the invasion capacityof trophoblast is largely unknown. In this project, we’ll apply the emerging techniqueof micro RNA (miRNA) to understand how miRNA regulate the TRI-mediatedtrophoblast invasion. Using miRNA array, we hope we can identify the newregulators of trophoblast invasion, and then using them as biomarkers to early detectpreeclampsia.In the first year, we use 3 different trophoblastic cell lines and the primarytrophoblast from the first-trimester curettage. We will clone 6 sets of RNAi targetingto TRI into the lentiviral plasmid, then deliver into the target cell. These RNAi mayresult in increasing invasion and migration of the trophoblast. After confirming byreal-time PCR and Western blotting that TRI is really decrease, we extract totalRNA from the cells then sent for miRNA analysis. The most significant upregulatedand downregulated miRNA (before and after treatment) will be picked up bybioinformatics. In the project of the second year, we overexpress the target miRNAsby plasmid transfection, and also knockdown them using miRNA sponge in the other.After confirmation by phenotypic and functional assays by invasion chamber andtime-lapse video microscopy, we can verify these miRNAs do regulate theTRI-mediated trophoblast invasion.In the last year, we’ll extract total RNA from the blood of the patients frompreeclamptic pregnancies and healthy control before the day of delivery. The samplesare sent to real-time PCR to compare the expressions of our target miRNAsidentified in the first 2 years. After statistics, the most significant changes of miRNAwill be picked up. They may serve the biomarkers of preeclampsia and probably leadto the development of the personalized treatment.