DnaJA3a基因在斑馬魚胚胎時期血管發育之研究

TMUIR > College of Medicine > Graduate Institute of Medical Sciences > Dissertation/Thesis > Item 987654321/58378

Please use this identifier to cite or link to this item: http://libir.tmu.edu.tw/handle/987654321/58378

Title:	DnaJA3a基因在斑馬魚胚胎時期血管發育之研究 Functional characterization of DnaJA3a gene during zebrafish embryonic vascular development
Authors:	劉詩敏 Lau, Sze-Man
Contributors:	醫學科學研究所周志銘
Keywords:	斑馬魚;胚胎發育;血管發育 zebrafish;embryonic development;vascular development;DnaJA3a
Date:	2018-12-18
Issue Date:	2020-01-07 13:33:03 (UTC+8)
Abstract:	DnaJ家族蛋白 (Heat shock protein 40 family) 是協助蛋白折疊的重要分子，近年來在許多研究中發現其家族蛋白在腫瘤的抑制與細胞凋亡過程中也扮演相當重要的角色。人類hTid1 (Tumorous imaginal discs 1) 又稱為 DnaJA3，是果蠅抑癌基因 (tumor suppressor gene) Tid56的同源基因。hTid1基因會在全身組織中表達，透過選擇性剪接可轉譯出 hTid1-L 和 hTid1-S 兩種蛋白產物，此兩蛋白在細胞內訊息傳遞路徑的功能主要為調節子 (regulator)，hTid1-L 主要功能為促進細胞走向細胞凋亡，而 hTid1-S 則為抑制細胞凋亡過程。由於兩種hTid1蛋白產物的C端胺基酸序列具有較大差異，hTid1-S較hTid1-L更具有停留於粒線體之特質。在斑馬魚中亦具有兩個hTid1旁系同源基因，命名為 DnaJA3a 和 DnaJA3b。在實驗室先前的研究發現，DnaJA3a基因無論在胚胎時期及成熟組織中其表現量一致且非常穩定，而DnaJA3b基因僅在受精後 72 和 96 小時 (hpf, hours post fertilization) 及大腦與肌肉組織中有較高的表現。為瞭解 DnaJA3a 在斑馬魚胚胎發育時期可能的功能，本研究先確認 DnaJA3a mRNA 在 24 到 48 hpf 有較高的表達，且主要表達在尾部中胚層與後端血島區域。當利用morpholino oligonucleotides (MO) 抑制 DnaJA3a 基因功能時，結果顯示在 24 hpf 與 48 hpf 胚胎之節間血管形成及發育受到明顯的抑制，而同時注射 DnaJA3a MO 與 DnaJA3a mRNA 則可以回復節間血管之正常發育。透過原位雜交 (WISH, whole mount in situ hybridization) 試驗亦發現 DnaJA3a MO 胚胎的節間血管 (ISV, intersegmental vessels)、主動脈 (DA, dorsal aorta) 與尾靜脈 (PCV, posterior cardinal vein) 發育明顯受到抑制。由於 vegf 與 Notch 訊息傳遞路徑為胚胎血管發育中重要的調控路徑，利用vegf與Notch路徑抑制劑 SU5416 及 DAPT 處理胚胎後發現其血管發育受抑制的表型與 DnaJA3a MO處理胚胎非常相似。而以抑制劑處理後結果顯示胚胎中的 DnaJA3a 表達受到抑制且下降。同時分析 DnaJA3a MO 處理後胚胎中參與血管發育相關分子表達之變化，結果顯示，24 hpf 除了 vegfAa與 vegfC 表達不受抑制外，其他相關分子表達量皆明顯減少；而 MO 處理 48 hpf 則參與血管發育相關分子表達皆減少。而過量表達DnaJA3a mRNA後，在 10 hpf 時加入抑制劑處理證實 DnaJA3a在胚胎血管發育是作用於 vegf 路徑之下游，並在 Notch 路徑之上游。因此證明了 DnaJA3a 確實會參與胚胎時期的血管發育，並可能透過 vegf 與 Notch 路徑調控血管發育。此研究成果，可提供 hTid1 (DnaJA3) 在癌症發展過程中，對於血管新生可能的分子調控機轉提供重要的資訊。 DnaJ family (Heat shock protein 40 family) involved in protein folding, recent researches have explored this family also involved tumor inhibition and process of cell apoptosis. Human Tid1 (Tumorous imaginal discs 1) also named as DnaJA3 is a homologue of Drosophila tumor suppressor gene Tid56. hTid1 expresses in whole tissues in human body and encodes two alternative splice variants, hTid1-L and hTid1-S. However, these isoforms exhibit opposing biological activities in response to cytotoxic insults. The hTid1-L promotes apoptosis whereas hTid1-S suppresses the process. Zebrafish (Danio rerio) also possess two DnaJA3 paralogues, named DnaJA3a and DnaJA3b. In our previous study showed the expression of DnaJA3a mRNA was ubiquitously during the developmental stages and adult tissues, whereas the expression of DnaJA3b mRNA was mainly at 72 and 96 hpf developmental stages and adult brain and muscle. Therefore, to identify the possible functions of zebrafish DnaJA3a, we indicated DnaJA3a mRNA is highly expressed in posterior blood island and tail mesoderm at 24 to 48 hpf. After that we performed with in vivo loss of function of DnaJA3a by morpholino, the result showed knockdown DnaJA3a leads to vasculature abnormalities in intersegmental vessels (ISV) at 24 and 48 hpf. Co-injection of DnaJA3a mRNA with DnaJA3a MO rescued absence of ISV. Meanwhile, DnaJA3a morphants regulated development of dorsal aorta (DA), posterior cardinal vein (PCV) and ISV as validated by whole mount in situ hybridization (WISH). Vegf and Notch signaling pathways are involved in embryonic vascular development. Thus, we sought to investigate the relationship between DnaJA3a, vegf and Notch signaling during embryonic vascular development. The data showed embryos treated with vegf signaling inhibitor (SU5416) and Notch signaling inhibitor (DAPT) at 26 hpf were analyzed for effects of ISV defected, moreover SU5416 and DAPT treatment reduced DnaJA3a expression. Finally, knockdown DnaJA3a caused down regulated of vegf and Notch signaling pathway molecules at 24 hpf expect vegfAa and vegfC, however all expression of pathway molecules was reduced at 48 hpf. Finally, overexpression of DnaJA3a by injection of DnaJA3a mRNA and treated with SU5416 or DAPT two inhibitors showed DnaJA3a acts in downstream of vegf signaling and upstream of Notch signaling. In summary, DnaJA3a might through vegf and Notch signaling to regulating zebrafish embryonic vascular development. The research results also provide a new direction of study on possible regulatory mechanisms of hTid1 (DnaJA3) in inhibition of angiogenesis in cancer progression.
Description:	碩士指導教授：周志銘委員：羅正汎委員：陳奕平
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Medical Sciences] Dissertation/Thesis

Files in This Item:

File	Description	Size	Format
index.html		0Kb	HTML	96	View/Open

View Licence

Loading...