建立人類子宮胎盤循環--侵襲性滋養層細胞角色之探討

Abstract

胚胎的成功著床，端賴絨毛外部滋養層細胞的精細調控之侵襲行為，然而目前對侵襲性滋養層細胞的分化、與其特化後的侵襲特徵，目前並無相關研究；而於懷孕中，滋養層細胞對蛻膜血管的重塑，其中所扮演的角色，目前的瞭解亦極為有限。本論文的前半部，吾人收集第一及第三周產期懷孕蛻膜的採樣，予以冷凍切片並免疫染色，同時進行廣泛的觀察這些侵襲性滋養層細胞在不同的時間、位置的型態，並對其特化表徵進行分析。 吾人發現，單極、紡錘型的滋養層細胞廣泛存在於母體蛻膜區，而其分佈密度則隨此侵襲路徑的深度而遞減，且其主要的細胞突起亦隨而變短。特別的是，於懷孕晚期，侵入子宮肌層淺部的滋養層細胞，會特化成星狀構造，而此星狀細胞亦可在轉型的蛻膜動脈週圍及動脈本身的平滑肌層中發現，而此星狀細胞的存在也會讓周邊的actin纖維排列變混亂、或產生空泡化的現象。吾人由觀察推論，星狀結構的特化可解釋滋養層細胞的控制式侵襲行為，並在蛻膜血管重塑的複雜細胞或生化過程中，扮演上游誘導之角色。 吾人第二步的實驗，在建立滋養層細胞的活細胞運動體外觀察模式。我們使用SV40特化的早期懷孕滋養層細胞株，將其培養於transwell invasion chamber的上層，再於6, 24, 48, 72小時不同的時間點，將留於上層及侵入下層的細胞作逐一分析，計算其TGF-beta第一型受體之陽性比例。結果發現滋養層細胞的侵襲，在24小時已達最高峰；上層細胞的TGF-beta第一型受體陽性表現比例有明顯隨時間遞減的趨勢，且下層細胞(代表完成侵襲的細胞)其TGF-beta第一型受體的表現，則自一開始即明顯發生向下調控的情形， 代表低TGF-beta第一型受體的表現與高滋養層細胞的侵襲能力有相關。為證實這樣的論點，我們在transwell上層細胞加入TGF-beta第一型受體的專一抑制劑(SB431542)或TGF-beta3因子來共同培養，結果發現抑制TGF-beta第一型受體確可明顯促進滋養層細胞侵襲的能力，而TGF-beta3因子的效果則全然相反，且此促進或抑制的效果均呈明顯地劑量相關之影響。 在進一步的實驗中，我們將細胞培養於培養皿中，並以活細胞觀察儀觀察實驗組(加入SB431542或TGF-beta3因子)及控制組之細胞動態變化，並在24小時實驗終了時，將細胞影像做整合型態分析。結果顯示，加入SB431542會使細胞面積變小，型態趨向紡錘細胞，吾人推測此為促使滋養層細胞使有利侵襲之重要因素；而TGF-beta3造成的效果則完全相反。另動態分析也顯示，加入SB431542使細胞於培養皿中的移動速度減慢，但明顯增加移動的平均角向量，而依細胞運動的路徑來看，這些高平均角向量的細胞均有較一致的移動方向，吾人推測如此更有利於滋養層細胞穿破細胞間或細胞與間質間的藩籬；而wound healing assay亦獲得類似結果。另由活體蛻膜切片觀察，發現滋養層細胞在細胞柱(滋養層細胞的不活動區)，其TGF-beta第一型受體表現均向上調控，而於蛻膜處(滋養層細胞的侵襲區)則明顯向下調控。吾人結論為，TGF-beta第一型受體的向下調控，可能是造成滋養層細胞脫離細胞柱、並開始向蛻膜侵襲的一個重要因子。

Successful implantation of embryo relies on the delicate control of invasion for extravillous trophoblasts. However, current studies on the phenotypic differentiations of invasion trophoblast and their relevant motile behaviors are still lacking. In addition, the precise role of invasive trophoblast in decidual vascular remodeling is not well-understood, either. In the first part of this dissertation, we prospectively collected the decidual sampling of the first and third trimester pregnancy, and processed with cryostat sectioning and immunohistochemistry. The aim of observation is to determine the phenotypic differentiations of invasive trophoblasts at different times and location, and postulate the inference for the relevant behavior of their specialized transformation. After extensively inspection of decidual specimens containing invasive trophoblasts and vessels, we found the unipolar, spindle-shape trophoblasts extensively infiltrated in maternal decidual stroma. The density of distribution, however, presented an inverse proportion with the depth of invasive pathway. Interestingly, those trophoblasts invading into myometrium at the third trimester transformed into stellate architecture. Meanwhile, stellate trophoblasts also existed in the surroundings of decidual spiral arteries, or even penetrated and harbored inside the tunica media of the vessel. Besides, the existence of stellate trophoblasts also perturbed the arrangement of actin fibers, or resulted in clear lacuna inside the tunica media. From these observed data, we suggested that the stellate transformation might explain the controlled invasion of invasive trophoblasts during pregnancy, and probably play an upstream role in the initiating the decidual vascular remodeling. My second experiment is to establish the in vitro model of living trophoblast observation by time-lapse video microscopy. First we used the cell line from SV40-transformed trophoblasts from first-trimester pregnancy for the transwell invasion assay. We seeded the cells to the upper chamber of transwell, and observed the cells staying in the upper or invading into lower chambers at 6, 24, 48, 72 hours. We found that cell invasion into lower chamber reached a plateau at 24 hour, and there was a trend of type I receptor of TGF beta (TbRI) downregulation for cells in upper chamber chronologically. Most importantly, TbRI expression in trophoblasts at lower chamber (representing invasive cells) remained low throughout the studied period. The data indicated that lower TbRI expression was associated with higher invasion capacity of trophoblast. To prove this notion, we further added selective TbRI inhibitor (SB431542) or TGF-beta3 in the trophoblast culture at upper chamber. The results yielded clear evidence that inhibited TbRI expression did increase the invasion ability of trophoblasts, while TGF-beta 3 supplement had an inhibitory effect of invasion, on the contrary. Besides, both the TbRI inhibitor and TGF-beta3 demonstrated a dose-related effect on invasion. The trophoblast culture was further evaluated with cine images recording by time-lapse video microscopy. We conducted 3 groups of experiment – supplemented with TbRI inhibitor or TGF-beta3 as experiment groups, and normal culture condition served as control group. At the end of 24-hour culture, we snapshotted the cell image and analyzed by routine of integrated morphometric analysis provided by Metamorph software. The result showed treatment with TbRI inhibitor significantly decreased the cellular area and dragged the cell into spindle shape, whereas the effects of TGF-beta3 were completely opposite. In addition, motion analysis also demonstrated that treatment with TbRI inhibitor significantly increased the angular vector of trophoblast motion but decreased migration velocity. Tracing the migration path of those with high angular vector, a persistent directionality of motion can be consistently found. We postulated it could promote the ability of trophoblast to penetrate membranous pore of transwell or the cell-cell or cell-matrix along the invasive pathway in vivo. We also investigated the in vivo specimen. Those trophoblasts in cell column demonstrated TbRI up-regulation, while trophoblasts in the interstitial area downregulated their TbRI expression. We concluded that, downregulation of TbRI expression is probably an important factor that induces the trophoblast detached cell column and start to invade maternal decidual stroma.