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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48525

Title: akirin2 在斑馬魚肝臟成長及病理發生上之角色與機制研究
Roles and molecular mechanisms of akirin2 in liver growth and pathogenesis of zebrafish
Authors: Yun-Lung Wang
王韵倫
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 斑馬魚;肝臟
Akirin2;liver;zebrafish
Date: 2015
Issue Date: 2018-08-22T06:05:44Z
Abstract: Akirins為一種缺乏DNA-binding domain核蛋白,除了鳥類以外,在大多數脊椎動物上有兩個Akirin基因:Akirin1及Akirin2。兩者皆利用和其他核蛋白結合參與諸多訊息傳遞路徑調控。已知Akirin1功能為藉由活化Helix-loop-helix肌肉調節轉錄因子使骨骼肌生長,當不受Myostatin抑制或過量表現時,會造成肌肉肥大。Akirin2則目前主要被發現參與免疫反應及癌症發生過程。免疫方面,抑制老鼠B-cell表現Akirin2將減少其數目且阻礙免疫反應。而在部分癌症細胞株中,Akirin2和14-3-3β組成細胞核內複合體並活化mitogen-activated protein kinase (MAPK)訊息傳遞路徑,促進腫瘤生成和轉移。另外,先前本實驗室以Morpholino (MO)抑制胚胎轉譯Akirin2時,發現除各胚層廣泛異常外,肝臟有縮小現象。綜合上述,Akirin2皆與細胞增生相關。本研究使用高效率Tol2轉位子,搭配雙向TetOff表現系統及斑馬魚fabp10a之2.9 kb長肝臟專一性啟動子(LF2.9),建立肝臟專一性過量表現Akirin2的基因轉殖斑馬魚品系,以探討Akirin2對肝細胞增生和進一步是否癌化之功能及相關分子機制。H&E染石蠟切片顯示年齡3個月以降的基因轉殖成魚,其肝臟具細胞質組成改變與血液蓄積現象。接著利用微陣列生物晶片(Microarray)和Ingenuity Pathway Analysis(IPA)預測出4個月大基因轉殖成魚相較於對照組,Akirin2在肝臟過量表現時,主要調控細胞增生存活及脂質代謝、分子運輸有關訊息傳遞路徑。再以qPCR分析所有參與細胞增生存活之基因mRNA表現量,得出十個受Akirin2過量表現穩定調控的基因:dusp1、arnt、cd36、fgb、sod3a、lifra、il6st、jak1、stat3、igf2b。總結上述結果,雖然未觀察到細胞增生及癌症發生過程,但我們推測肝臟專一性過量表現Akirin2可能藉由Akirin2-14-3-3β複合體形成,抑制DUSP1表現以活化MAPK訊息傳遞路徑,下游再磷酸化HIF1α增加HIF1形成,並啟動抗缺氧及血管新生等諸多相關路徑。
A kind of nuclear protein, Akirins, of whom there are usually two types—Akirin1 and Akirin2—in most vertebrates except Aves. Both regulate many signal transduction pathway by interacting with other nuclear protein. Previous studies report that Akirin1 up-regulates the myogenesis regulatory factors, which possess a characteristic basic helix-loop-helix domain, to increase the growth of skeletal muscle. Overexpression of Akirin1 or loss of inhibition for Myostatin result in increased skeletal muscle mass referred to as “double muscling”. In contrast, Akirin2 had previously been shown to regulate immune response and progression of tumors. Inhibition of expression of Akirin2 in B-cells decreases the amount of them and interferes with immune response. In several tumor cell lines, Akirin2 and the phosphoserine-threonine-binding protein 14-3-3β form a complex in nucleus to activate the mitogen-activated protein kinase (MAPK) pathway so that tumorigenicity and metasis etc. are promoted. Additionally, we have found that when the expression of Akirin2 was suppressed by microinjecting the morpholino (MO), the larvae (120 hpf) grow and develop abnormally, and their liver seemed to lessen. Briefly, it seems that Akirin2 associates with cell proliferation. Thus, in this study, for researching the role of Akirin2 in signal transduction pathway participating in hepatocyte proliferation and, futher, carcinogenesis, we used Tol2 transposon system combined with TetOff regulatory system and 2.9 kb liver-specific promoter of fatty acid binding protein 10a (fabp10a) to establish a transgenic zebrafish line overexpressing akirin2 specifically in liver. We observed the change of cytosol and/or accumulation of blood in liver of transgenic zebrafish older than three months by H&E staining. By microarray and Ingenuity Pathway Analysis (IPA), we found that the genes may be regulated by specific overexpression of Akirin2 in liver, and some of them may involve the signal transduction pathways about cell-survival, metabolism of lipid and molecule transport. Then, using qPCR to confirm the all expression of genes concerning cell-survival, we found the ten genes: dusp1、arnt、cd36、fgb、sod3a、lifra、il6st、jak1、stat3、igf2b, which were regulated stably by overexpression of Akirin2. In summary, although we had seen no evidence of carcinogenesis, we presume that specific overexpression of Akirin2 in liver is a important role to beginning the pathways regarding hypoxia and angiogenesis.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010133044.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48525
Appears in Collections:[水產養殖學系] 博碩士論文

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