- 學位論文
人類絨毛外滋養層細胞分化的調控機制
Regulatory mechanism of human extravillous trophoblast differentiation
摘要
在人類胎盤發育的過程中，滋養層細胞 (trophoblast cell) 主要透過兩種分化途徑來形成完整的胎盤結構。其中一種是形成絨毛膜的絨毛，其內層是具有增生及分化能力的細胞性滋養層細胞 (cytotrophoblasts, CTBs) 所組成，並藉由細胞融合形成絨毛外層的多核融合滋養層細胞（syncytiotrophoblasts, STBs)，這對於胎兒與母親之間營養與氣體的交換十分重要。另一種是形成錨定絨毛 (anchoring villus) 並與蛻膜 (decidua) 接觸，其中的部分細胞性滋養層細胞可進一步分化為絨毛外滋養層細胞 (extravillous trophoblasts, EVTs)，它們可重塑母體的螺旋小動脈，提供足夠的血液供應給成長中的胎兒。由此可知滋養層細胞的分化與胎盤發育和妊娠相關疾病有很大的關聯，因此，透過了解滋養層細胞的分化機制，可以幫助我們在臨床上治療與妊娠相關疾病。 為了瞭解滋養層細胞的分化，我們從人類的胎盤中建立了由細胞性滋養層衍生的滋養層幹細胞 (trophoblast stem cell, TSC)，並成功地將其分化為融合滋養層細胞和絨毛外滋養層細胞，接著我們進一步分析這三種型態的滋養層細胞，從數據中發現到IFITM1 (interferon-induced transmembrane protein) 在絨毛外滋養層細胞中高度表達，在細胞株和人類的胎盤組織也確認IFITM1專一表達於絨毛外滋養層細胞中，而在先前的研究證明了IFITM1能夠抑制內源性逆轉錄病毒的融合蛋白合胞素 (syncytin)，來阻斷細胞的融合。因此，我們認為IFITM1是能夠影響滋養層細胞分化的重要角色之一。而從胎盤的結構中我們可以發現絨毛中與蛻膜接觸的細胞性滋養層細胞才會分化成絨毛外滋養層細胞，是否代表蛻膜細胞能夠影響滋養層細胞的分化命運，所以我們透過共培養系統，觀察到蛻膜細胞能使滋養層細胞的侵襲能力增加，而在處理蛻膜細胞條件培養液的實驗中也發現到IFITM1及和絨毛外滋養層細胞分化相關的基因表現量上升，而對融合滋養層細胞所需的合胞素則沒有顯著差異，所以我們認為蛻膜細胞能藉由某些因子誘導滋養層細胞往絨毛外滋養層細胞分化。 為了進一步證明IFITM1會影響到滋養層細胞的分化，我們首先將IFITM1過表現於滋養層幹細胞再進行分化，卻發現對於融合滋養層細胞的細胞融合能力以及分化為絨毛外滋養層細胞的進程沒有顯著影響，而若是把IFITM1利用RNA干擾的方式下調，則發現到IFITM1的缺失會影響到滋養層幹細胞分化成絨毛外滋養細胞，其中也包含了和絨毛外滋養層細胞分化的相關基因表現量減少，除此之外，我們發現到當抑制TGFβ時能使IFITM1的表現量上調，且在絨毛外滋養層細胞分化的早期便能觀察到IFITM1表達出現。綜上所述，我們的研究表明了蛻膜細胞分泌有關抑制TGFβ信號傳導的因子，而誘導IFITM1高度表達，使滋養層細胞往絨毛外滋養細胞的方向分化。
並列摘要
During human placental development, trophoblast cells differentiate through two major pathways to establish the dynamic placental structure. One is the formation of chorionic villus which is composed of an outer layer of multinucleated syncytiotrophoblasts (STBs) and inner stem cell-like cytotrophoblasts (CTBs). Indeed, CTBs undergo cell-cell fusion to form STBs that are essential for nutrient-gas exchange between fetus and mother. The other is the formation of anchoring villus which attaches to the decidua and from where CTBs proliferate and differentiate into extravillous trophoblasts (EVTs) to remodel maternal spiral arterioles and provide sufficient blood supply to the growing fetus. These differentiation pathways are crucial for placental development, and abnormalities in these pathways are associated with pregnancy disorders. Therefore, investigation on the mechanism of trophoblast differentiation is important for the development of clinical treatments of pregnancy disorders. To better understand trophoblast differentiation, we have established the CTB-derived trophoblast stem cells (TSCs) from the human placenta and these TSCs can successfully differentiate into STBs and EVTs. Gene expression profiling of TSCs, STBs, and EVTs shows that interferon-induced transmembrane protein (IFITM1) is highly expressed in EVTs. We further demonstrate that IFITM1 is primarily expressed in EVTs in vitro and in vivo. Previous studies have indicated that IFITM1 blocks cell fusion mediated by the endogenous retroviral fusogens, syncytins. Hence, we hypothesize that IFITM1 is a key factor in regulation of trophoblast differentiation. Because CTBs encounter decidual cells when they are differentiating into EVTs, we also hypothesize that decidual cells may modulate EVTs differentiation. Indeed, we detect increased invasive ability of TSCs when co-cultured with decidual cells. Moreover, treatment of TSCs with the conditioned medium of decidual cells upregulate expression of IFITM1 and genes related to EVT differentiation, but not syncytin. To study how IFITM1 regulates trophoblast differentiation, we overexpress IFITM1 in TSCs and test their capacities in STB and EVT differentiation. No significant effect of IFITM1 overexpression was detected on the cell-cell fusion in STB differentiation and the expression of EVT markers. Interestingly, knocking down IFITM1 by RNA interference significantly suppresses EVT differentiation in terms of marker expression and cell morphology. We further demonstrate that repression of TGFβ signaling pathway in TSCs leads to IFITM1 upregulation and that IFITM1 expression is an early event in the differentiation process of EVTs. Taken together, our study indicates that activation of IFITM1 expression promotes the differentiation of TSCs into EVTs via inhibition of TGFβ signaling, which can be contributed by decidual factors.