2017-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/653442摘要：有效的滋養層細胞侵襲，對早期懷孕成功著床佔著最關鍵的因素。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。如何使用體外模式，去正確評估誘發侵襲不足或過度的各種因子，對開發疾病治療及預防的對策，有舉足輕重的重要性。目前探討滋養層細胞侵襲的文獻雖然很多，但使用的幾乎都是單細胞層的二維培養模組。根據腫瘤細胞侵襲的研究，三維培養模式比較接近體內的生理狀況，而二維培養模式雖然容易操作，但其侵襲研究的結果，有時甚至與三維模組完全相反。在本計劃，我們使用3A-Sub E 滋養層細胞株，先使用傳統的單層培養法放大，當細胞量足夠後，將細胞打散離心，加入培養液調至細胞濃度9 x 103 cells/30μl。利用表面張力將此細胞培養液置于一100 mm 培養皿上蓋內面製成懸滴，一個上蓋大約可放入40 個含細胞株之懸滴，將培養皿中加入5ml PBS，然後翻轉此上蓋，蓋到培養皿上，放入培養箱中培養72 小時，利用重力等待滋養層細胞球體形成。另外於96 孔培養皿中，加入Matrigel 與血清1:1 當底層，將形成的滋養層細胞球體清洗後放入培養皿中，再加入TGF1, 3, TGF 受體專一拮抗劑，以及TBR1 基因的短髮夾核糖核酸，分別培養在正常氧氣和1%缺氧環境中，去比較不同的TGF路徑的處理對滋養層細胞球體侵襲的影響。同樣的處置，亦用傳統2 維侵襲評估方式(轉孔遷移測定及傷口癒合測定)來做為比較。本計劃主要旨在建立一個容易實行、高通量、不需要複雜儀器與特殊藥品的三維滋養層細胞培養系統，以期對體外培養的滋養層細胞，可有最正確符合生理狀況的預測(尤其是早期著床環境的缺氧梯度)，以期發展預防或治療妊娠合併症的對策。<br> Abstract: A successful trophoblast invasion plays a pivotal role in the pregnancy implantation. The twomajor causes of maternal death, preeclampsia and placenta accreta, are due to either shallow orexcessive trophoblast invasion. To develop their preventive strategy, it is important to investigatethose cues resulting in insufficient or undue trophoblast invasion. Although there are abundantliteratures in evaluation of trophoblast invasion, the majority of them used the 2D monolayer culture.According to the tumor invasion assay, three-dimensional cell culture model is more predictive andinformative for the in vivo environment. The 2D monolayer culture method, although easier tohandle, can sometimes present contrary results to those derived from 3D system.In this project, we use the trophoblastic cell line (3A-sub E) that derived from immortalizedfirst-trimester villi. The cells are cultured in monolayer to confluence, and then trypsinized,resuspended as usual. These cells are titrated with medium to the concentration of to 9103cells/30μl. We then remove the lid from a 100 mm tissue culture dish and place 5 ml of PBS in thebottom as a hydration chamber. We invert the lid and deposit 30 μl drops using a 100 μl pipettoronto the bottom of the lid. The lid is inverted to cover the petri dish, and then incubate for 72 hoursto let trophoblast spheroids grow using gravity. Those spheroids are transferred to Matrigel-coatingchamber, and subjected to different treatment as well (such as normoxia and hypoxia environment,TGF1 and 3, selective inhibitor of type I TGF receptor or shTBR1).This project is to establish an easy-perform high-throughput 3D culture system of trophoblastspheroid without the need for sophisticated equipment and reagent. This culture system is expectedto be close to the in vivo environment, especially for hypoxia gradient occurring during earlyimplantation; and hopefully, can be applied in developing the strategy to prevent thosecomplications from inadequate trophoblast invasion.滋養層細胞侵襲懸滴培養球體立體培養缺氧轉化生長因子trophoblastinvasionhanging drop culturespheroid3D culturetransforming growth factor