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標題: | 水解磷酸酯受器3於斑馬魚的造血功能中扮演之角色 The Roles of Lysophosphatidic Acid Receptor 3 in Hematopoiesis of Zebrafish |
作者: | Ya-Chi Chang 張雅琪 |
指導教授: | 李心予 |
關鍵字: | 水解磷酸酯受器3,斑馬魚,造血功能,紅血球生成, Lysophosphatidic acid receptor 3 (Lpa3),zebrafish,hematopoiesis,erythropoiesis, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 造血功能是造血幹細胞進一步分化成特定血球族群的動態過程。目前已知水解磷酸酯受器3 (Lysophosphatidic acid receptor 3, LPA3) 參與造血幹細胞與骨髓微環境的調控。本實驗室先前的研究發現,在人類造血幹細胞與斑馬魚生物模式中,活化 LPA3 會提高紅血球生成素 (EPO) 誘導的紅血球生成。此外,在斑馬魚中利用核酸類似物 (morpholino, MO) 降低 LPA3 會導致紅血球生成的缺失。然而藥物刺激或是化學物引發的基因調控有可能引發額外的未知影響。因此,本研究希望在斑馬魚上使用基因剔除 (Knock-out) 的技術探討 LPA3 於造血功能中扮演的角色。本次研究中,我們使用 transcription activator-like (TAL) effector nucleases (TALEN) 在 LPA3 基因組的第一段內插子中踢除兩個鹼基對,建構出 LPA3 基因缺失的斑馬魚。和野生型基因相比之下,LPA3 踢除的胚胎所含的血紅素與游動能力並沒有顯著的差異。然而,在受精後的三天內的胚胎中,造血相關的基因表現出現了延遲的現象,顯示 LPA3 可能在斑馬魚胚胎發育中影響了造血的功能。此外,在 LPA3 基因踢除的成魚中,其造血中樞 - 全腎骨髓的面積縮小,且全腎骨髓中的血球細胞族群比例也產生變化。綜觀諸多結果顯示 LPA3 在斑馬魚的胚胎或成體時期的造血功能中都扮演很重要的角色。 Hematopoiesis is a dynamic process by which hematopoietic stem cells (HSCs) differentiate into the specific blood cell lineages. Lysophosphatidic acid (LPA) receptors have been shown to be involved in the regulation of hematopoiesis in HSCs and bone marrow microenvironment. In our previous studies, we demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoietin(EPO)-dependent erythropoiesis in HSCs and zebrafish. Moreover, knockdown of Lpa3 expression by using morpholino (MO) caused erythropoietic defects in zebrafish embryos. However, the pharmacological activation and chemical-dependent gene-depletion of LPA3 may have redundant effects during the development of zebrafish. Therefore, we aim to generate Lpa3 knockout zebrafish to clarify the roles of Lpa3 in hematopoiesis. In this study, we established LPA3 knockout (Lpa3-/-) zebrafish by deleting 2 base pairs in exon 1 using transcription activator-like effector nucleases (TALEN). As expected, the expressions of early hematopoietic markers are delayed in Lpa3-/- embryos within 3 days post fertilization (dpf) by real-time PCR, implying that Lpa3 is involved in the embryonic hematopoiesis in zebrafish. However, 5 dpf Lpa3-/- larvae showed no deficiency in the amount of hemoglobin and locomotor activities by histochemistry staining and high-throughput behavioral studies, respectively. Interestingly, adult Lpa3-/- zebrafish showed smaller whole kidney marrow (WKM) in histological analysis compared to the wild type. Flow cytometry analysis also showed the decreased population of precursors and lymphocytes in adult Lpa3-/- zebrafish, suggesting that Lpa3 may involve in the lymphopoiesis of zebrafish. Taken together, these findings reveal critical roles of Lpa3 in the embryonic development and adult hematopoiesis in zebrafish. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66937 |
DOI: | 10.6342/NTU201702592 |
全文授權: | 有償授權 |
顯示於系所單位: | 生命科學系 |
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