人類凝血酶調節素(thrombomodulin, TM)是一種廣泛表現在內皮細胞的第一型穿膜醣蛋白，在生理上扮演參與活化蛋白質C抗凝血路徑上的重要角色。然而在人類鮮少發現因為基因突變使TM功能降低的個體，暗示著TM缺失在胚胎發育過程中可能會導致胚胎死亡。過去的研究指出，同源性TM基因剔除小鼠在胎盤發育至第8.5天時即出現無法發育而死亡的現象。更進一步的研究顯示剔除TM影響胎盤巨滋胚層母細胞的成熟及導致分化的滋胚層細胞死亡，最終影響到母體與胚胎間血液的交換而導致胚胎缺乏養分致死。利用胎盤特異TM剔除技術產生的TM剔除小鼠可以在母鼠子宮內存活超過8.5天，但是在第16.5天以前仍然造成胚胎死亡的現象，這個時期小鼠的死因是由於致死的耗損性凝血病變。相反的，蛋白質C剔除的小鼠卻可以正常發育且出生，暗示著胚胎正常發育是不需要經由蛋白質C參與的抗凝血路徑。這些觀察表示TM在胚胎發育時期除了參與蛋白質C的抗凝血路徑之外，還扮演著其他更重要的角色。目前科學家雖然已建立了許多TM突變及組織特異性剔除TM動物模式，TM剔除小鼠發生死亡之前器官發育的外觀上似乎正常，但是TM在胚胎發育過程中扮演的角色尚未清楚。在本篇研究，我們期望利用斑馬魚當做動物研究模式，探討TM在脊椎動物早期胚胎發育中所扮演的角色。由於斑馬魚有行體外受精、胚胎個體可以不依賴母體提供養分而獨立的發育的特點，提供一個可排除掉胎盤對於胚胎影響的動物模式。我們利用RT-PCR來偵測斑馬魚TM在胚胎發育時期表現的時間，發現斑馬魚TM由胚胎發育至受精後8小時(hours post fertilization, hpf)時期開始微量出現，由10 hpf至12 hpf表現量漸漸增加，自12 hpf到96 hpf都持續大量表現。利用嗎啉基反義代寡核苷酸來降低斑馬魚TM蛋白質的產生，延遲胚胎原腸胚時期的外包作用，而在胚胎心臟開始跳動後發現其圍心膜腫大與心跳、血液循環變慢的症狀與可能影響到血管的通透性。此外，這些胚胎在原腸胚時期的存活率隨著嗎啉基劑量的增加而下降。總結來說，由本論文所觀察到的現象顯示TM在胚胎發育的過程中，可能對於心血管系統功能的健全具有一定的影響力。

Thrombomodulin (TM) is a type I transmembrane glycoprotein receptor and widely expressed on the surface of endothelial cells. It involves in the protein C mediated anticoagulant pathway. So far, there was no report indicated almost or complete deficiency of TM in humans. It implied that loss of TM may cause death during embryogenesis. Some studies showed that homologous knockout of TM in mice caused embryonic lethality at day 8.5 post coitum. Further study indicated that abortion of TM knockout mouse is growth arrest of immature trophoblast cells and at the same time the elicitation of apoptotic cell death of differentiated trophoblast cells, ultimately causing embryonic lethality due to destroying the exchange of nutrient between fetal and maternal circulation in the placenta. Otherwise, TM-deficient mice which had tissue-restricted expression of TM in the placenta can survive beyond 8.5 post coitum, but encountered a secondary development block and died before 16.5 post coitum owing to lethal consumptive coagulopathy. In contrast, protein C deficient mice presented normal embryonic development and survived after birth. It implies that the protein C mediated anticoagulant pathway is not required for embryonic development. These observations also suggest that TM may serve an essential developmental function not only involving in the initiation of the protein C mediated anticoagulant pathway. So far, a series of mouse strains with TM mutation and conditional knockout was available. However, TM knockout embryos showed normal organogenesis before lethality, the biological function of TM in embryonic development remains largely unclear. In this study we aimed at investigating the roles of TM in early vertebrate embryogenesis by using the zebrafish model. The features of using zebrafish as the experimental model included its ex utero development and growth up without the nutrient of maternal supplies. It is suitable to study function of TM in early embryogenesis and the effect of TM function in the placenta can also be excluded. At first, we explored the gene expression patterns and functions of zebrafish TM (zTM) in embryonic development. The results showed that zTM expression can be detected at the 8-9 hours post-fertilization (hpf) and strongly expression at the 12-96 hpf. Knocking down of zTM by injecting morpholino antisense oligonucleotides yielded epiboly delay during gastrulation. After heart beating, stringent knockdown of TM resulted in abnormal cardiac morphology with edema in pericardial sac in a dose-dependent manner, decrease of heart beat, deteriorated blood circulation, and abnormal vessel permeability. Moreover, the survival rate of zTM knockdown embryos also showed a dose-dependent decrease during gastrula period. Taken together, TM expression is involved in the cardiovascular cardiac function during embryonic development in zebrafish.

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