臺灣博碩士論文加值系統

水解磷酸脂(Lysophosphatidic acid, LPA)為一脂溶性小分子，其調控許多生理機制，如神經細胞的分化、癌症細胞的複製、轉移或存亡等等。水解磷酸脂會和水解磷酸脂受器1-6結合，引發下游的訊息傳遞路徑。動物體內全部的血球，包括紅血球、淋巴球和血小板，皆由造血幹細胞(Hematopoietic Stem Cells)分化而來。在先前的研究中，我們已經瞭解造血幹細胞會表現水解磷酸脂受器，並且水解磷酸脂受器3會促進紅血球的分化。但其餘的水解磷酸脂受器對於血球分化的影響尚未清楚。而在分化的過程中，紅血球和血小板擁有共同的前驅細胞。因此本研究利用斑馬魚來探討水解磷酸脂受器2是否會同時調控紅血球及血小板的分化路徑。我們在斑馬魚胚胎注射抑制基因表現分子後的第四天，用紅血球染劑染色；同時利用real-time PCR測定紅血球相關基因(Hbae1和GATA1)及血小板基因(CD41)的mRNA表現量。接著在基因轉殖魚Tg(eGFP:CD41)胚胎內注射抑制基因表現分子藉以去觀察血小板生成。實驗結果發現，注射抑制基因表現分子後，血紅素有增多的趨勢；Hbae1,GATA1和CD41表現量也上升。在Tg(eGFP:CD41)胚胎注射抑制基因表現分子後，觀察到的GFP細胞數量也呈現上升。另外，將斑馬魚胚胎以水解磷酸脂受器2促進劑處理後再用紅血球染劑與real-time PCR進行分析。實驗結果發現，水解磷酸脂受器2促進劑會使斑馬魚的紅血球數量下降；Hbae1及CD41的表現量也降低。研究結果顯示，水解磷酸脂受器2 會同時抑制紅血球和血小板的分化。

Lysophosphatidic acid (LPA) is a small lysophospholipid which regulates many cell behaviors, such as cell proliferation, migration, and survival. LPA binds to a family of G-protein-coupled receptor, LPA receptor 1-6, and activates its downstream pathways. Hematopoiesis is a developmental process that hematopoietic stem cells (HSCs) differentiate into all types of blood cells, including erythrocytes, lymphocytes, and megakaryocytes. In our previous study, we demonstrated that activation of LPA3 could enhance erythropoiesis processes. However, the roles of other LPA receptors in hematopoiesis remain unclear. Since erythrocyte and megakaryocyte share the common progenitor cell, we attempted to investigate the role of LPA receptors in megakaryopoiesis processes. To clarify the role of LPA2 in erythrocyte and megakaryocyte differentiation in zebrafish, we first injected anti-sense morpholino oligonucleotide of LPA2 into wild-type zebrafish embryos at one-cell-stage. O-dianisidine staining for hemoglobin and real-time PCR were used to determine the expression of erythrocyte and mRNA expression of erythropoietic markers, Hbae1 and GATA1, or megakaryocyte marker, CD41, respectively. Moreover, we also injected LPA2 morpholino into embryos of Tg(eGFP:CD41) transgenic line. Our results demonstrated that knockdown of LPA2 enhanced the staining at CHT and mRNA levels of Hbea1, GATA1, and CD41. In addition, the number of megakaryocyte in Tg(eGFP:CD41) zebrafish embryos was also increased. Furthermore, we performed pharmacological experiment to confirm these results by incubating embryos with the LPA2 agonist GRI977143 and RP-239. Results from staining of o-dianisidine and real-time PCR of Hbea1, and CD41 showed that both protein and mRNA levels were suppressed by LPA2 agonists. In conclusion, our results suggested that activation of LPA2 inhibit differentiation of erythrocyte and megakaryocyte in zebrafish.

口試委員審定書………………………………………………………………………………………………………………………i
致謝…………………………………………………………………………………………………………………………………………ii
Catalog………………………………………………………………………………………………………………………………iii
摘要……………………………………………………………………………………………………………………………………………v
Abstract………………………………………………………………………………………………………………………………vi
Introduction……………………………………………………………………………………………………………………1
Hematopoiesis in vertebrate…………………………………………………………………1
Erythropoiesis in vertebrate………………………………………………………………3
Megakaryopoiesis in vertebrate…………………………………………………………4
Zebrafish hematopoiesis……………………………………………………………………………5
Function of LPA and LPA receptors…………………………………………………7
Materials and Methods……………………………………………………………………………………………10
Zebrafish maintenance and embryos collection……………………10
Antisense morpholino injection…………………………………………………………10
Agonists Treatment…………………………………………………………………………………………11
O-dianisidine staining………………………………………………………………………………11
Total mRNA extraction and real-time PCR…………………………………11
Fluorescence microscope……………………………………………………………………………13
Results…………………………………………………………………………………………………………………………………14
Knockdown of LPA2 by morpholino injection increases erythropoiesis………………………………………………………………………………………………………………14
LPA2 agonists inhibit hemoglobin expression………………………15
LPA2 inhibits thrombocyte differentiation……………………………16
Discussion…………………………………………………………………………………………………………………………19
Reference……………………………………………………………………………………………………………………………22
Figures…………………………………………………………………………………………………………………………………32

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