2015-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/671700摘要：水解磷酸脂是一種結構簡單的磷脂質小分子，藉活化不同類型之水解磷酸脂受器來調控細胞內諸多生理作用。本實驗室先前研究發現水解磷酸脂受器３參與在紅血球分化調控之機轉。於初步實驗結果中顯示，除了紅血球生成之外，水解磷酸脂受器２／３也在血小板分化的過程中扮演重要角色。在造血幹細胞分化過程中，紅血球與血小板於分化上游有共同前驅細胞，經分化後衍生出兩個血球分支。人類血癌細胞株K562 細胞於適當刺激下，可表達出人類巨核細胞標記基因，因而可被視為巨核細胞分化之研究模式。在施加水解磷酸脂受器２促進劑後，經由開啟貝塔-連環蛋白的訊息傳遞路徑來抑制造成血小板分化；而在施加水解磷酸脂受器３促進劑之下，經由增加活性氧物種和其相關之機轉，造成血小板分化之增強。此外，以核糖核酸干擾抑制水解磷酸脂受器２表達後，血小板分化基因表現上升；而抑制水解磷酸脂受器３之後，血小板分化基因表現下降。這些結果顯示，水解磷酸脂受器２與３對血小板分化具有拮抗的調控作用。在此延續之計劃中，我們預期於人類胚胎幹細胞以及K562 細胞中，同時過度表現和抑制不同的水解磷酸脂受器基因，以檢視血小板分化過程。在動物實驗方面，預計以血小板表現綠螢光蛋白之基因轉殖斑馬魚與水解磷酸脂受器基因剔除斑馬魚，進一步觀察血小板生成所受到之影響。同時也將用正常的小鼠和水解磷酸脂受器２／３剔除小鼠作為基礎，觀察水解磷酸脂受器之促進劑對於血小板分化和凝血功能之影響。這些研究結果應可對血小板相關疾病治療提供重要背景知識。<br> Abstract: Lysophosphatidic acid (LPA), a low molecular weight lipid growth factor, regulates numerous physiological functions through activating various G protein coupled receptors, LPA1-6. Our previous studies demonstrated that LPA activate erythropoiesis process through activating LPA3 in human hematopoietic stem cells (hHSCs) and zebrafish model. Furthermore, we also recently reported that LPA plays important roles during megakaryopoiesis, which is the differentiation process for platelet development, in K562 human leukemia cell line model. By using K562 cells, we found that activation of LPA2 and β-catenin-dependent pathway with LPA2 agonist, DMP, inhibited the expression of megakaryopoietic markers. On the other hand, LPA3 agonist, OMPT, enhanced megakaryopoiesis through activating ROS-dependent pathways. In addition, LPA2 shRNA treatment enhanced the expression of megakaryopoietic markers, while LPA3 shRNA treatment decreased their expression. Our data strongly indicated that LPA2 and LPA3 plays antagonized role during megakaryocyte differentiation through different signaling pathways. In this proposal, we attempt to overexpress and knockdown different LPA receptors simultaneously in K562 and hHSC models to monitor the platelet differentiation in vitro. Also, the functions of LPA2 and LPA3 signaling will also be confirmed in hHSC model by DMP and OMPT treatment respectively. Furthermore, we will also confirm our hypothesis by CD41-GFP and LPA2/3 TALEN transgenic zebrafish to investigate the roles of LPA2 and LPA3 in vivo. Finally, we will use different available pharmacological drugs to treat wild-type mice and LPA2/3-knockout mice to further investigate the roles of LPA receptors in platelet development and function. These results should clarify the roles of LPA receptors during megakaryopoiesis processes and provide important background information for development of potential treatment for diseases such as megakaryopenia.