吳金洌臺灣大學:漁業科學研究所林明轅Lin, Ming-YuanMing-YuanLin2010-05-102018-07-062010-05-102018-07-062007U0001-0511200717100800http://ntur.lib.ntu.edu.tw//handle/246246/181646IMP2 最早是在肝癌病人上的自體抗原發現，而IMP2 在肝癌發過程中，從慢性肝病像是脂肪肝，肝硬化到肝癌發生中間的機制，尚未清楚。到目前，並沒有任何IMP2 的轉基因動物。在本研究中，使用Tol2 跳躍子系統建立IMP2 轉基因斑馬魚，並進一步研究其分機制在肝癌發生過程。我從斑馬魚複製出IMP1 和IMP2 全長DNA。而斑馬魚的IMP1 和IMP2 在成魚肝臟表現量非常的低 。我用Tol2 跳躍子建立了由非洲爪蟾EF1α啟動子驅動的不同IMP2 表量轉基因魚。在2.5 個月大的IMP2 高表現量轉基因魚中，經由組切片發現了嚴重的脂肪肝。脂肪堆積在肝細胞的細胞質中，造成細核被推擠到邊緣。在IMP2 轉基因魚中，發現IGF2a 和IGF2b 會有別調控。而Akt2 在IMP2 高表現量轉基因魚中表現量增加。利用斑馬魚一萬四千點的微矩陣和Q-PCR 分析，發現脂肪合成酵素，脂酸合成脢( fatty acid synthase )、乙烯輔脢A 羧化脢( acetyl CoAarboxylase )、固醇輔脢A 去飽和脢-1 ( stearoyl CoA desaturase-1)，以與膽固醇生合成相關酵素的戊二酸輔脢A 合成脢( HMGCoAynthase )、戊二酸輔脢A 還原脢( HMGCoA reductase )、FPS 都被正控，而這些結果可能是因為脂肪關鍵轉錄因子SREBP1 活性被促。除了SREBP1，和脂肪合成相關的轉錄因子PPARγ和C/EBPα也IMP2 高表現量轉基因魚中都被正調控。而脂肪大量累積，對細胞成了內質網壓力(ER stress)，unfolded protein response 的關鍵轉錄因XBP1 和內質網chaperon 蛋白Hspa5/Grp78、Hyou1 和轉錄因子TF4 也都在高IMP2 表現量中被促進。這些結果顯示IMP2 會導致脂肝形成。過量表現IMP2 轉基因魚會經由Akt2 和脂肪相關轉錄因SREBP1、PPARγ和C/EBPα導致脂肪肝。IGF2 mRNA binding protein 2 (IMP2) was first identified as an auto-antigen p62, which was aberrantly expressed in the human hepatocellular carcinoma (HCC). The roles of IMP2 in HCC development, from chronic liver disease such as fatty liver, cirrhosis to HCC, are still poor understood. Up to now, no any animal model of IMP2 have been reported. In this study, I first intended to establish IMP2 transgenic zebrafish lines by Tol2 transposon-mdiated transgenesis to study the molecular mechanism of IMP2 during the progression of HCC. Full length IMP2 cDNAs were isolated from zebrafish by RT-PCR and RACE. Expression of endogenous IMP2 is rare in the normal liver of adult zebrafish. Transgenic zebrafish lines with different levels of IMP2 over-expression driven by Xenopus EF1a promoter were established. Severe fatty liver (steatosis) was observed in the liver of 2.5 month F1 transgenic zebrafish with high level IMP2 over-expression by HE staining. High accumulating lipid in the cytoplasm pushed the nucleus to the peripheral of the hepatocytes. Zebrafish IGF2a and IGF2b are differentially regulated by IMP2 in the liver of IMP-transgenic zebrafish. Zebrafish IGF2a expressed was activated in the liver of highly IMP2-overexpressed transgenic fish, whereas IGF2b was repressed by IMP2 in dose dependent manner. Expression of zebrafish Akt2 was increased in highly IMP2-overexpressed transgenic zebrafish. By using zebrafish microarray and Q-PCR analysis, enzymes involved in fatty acid synthesis such as acetyl CoA carboxylase, fatty acid synthase, stearoyl CoA desaturase-1, ELOVL1 and cholesterol synthesis ex. 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase were up-regulated in the liver of IMP2-overexpressed transgenic zebrafish. It may result from the increase of key lipogenic transcription factor SREBP1 expression. In addition to SREBP1, transcription factors PPARr and C/EBPa involved in lipogenesis were also up-regulated in the liver of highly IMP2-overexpressed transgenic zebrafish. Due to severe steatosis, ER stress response genes such as XBP1, a key transcriptional regulator of unfolded protein response (UPR); ER chaperones Hspa5/Grp78, Hyou1 and transcription factor ATF4 were strongly activated in the liver of high level IMP2 overexpressed transgenic zebrafish. These data suggest that IMP2 may play a role in fatty liver formation. Overexpression of IMP2 in liver of transgenic zebrafish leads to hepatic steatosis via activation of Akt2目錄文摘要-----------------1文摘要-----------------3、 前言------------------------5二型類胰島素生長因子訊息核醣核酸訊息結合蛋白(IMPs)--5肪肝-----------------------------------------9馬魚動物模式---------------------------------9究動機---------------------------------------10、 材料----------------------------------11、 實驗方法-------------------------------21、 實驗結果-------------------------------31馬魚IMPs基因全長選殖--------------------------31馬魚IMPs轉基因魚建立---------------------------32量表現斑馬魚IMP2導致肝臟累積脂肪---------------33量表現斑馬魚IMP2在斑馬魚肝臟會促進IGF2a表現-------34量表現斑馬魚IMP2促進脂肪合成相關轉錄因子和脂肪成關鍵酵素--------------------------------35量表現斑馬魚IMP2促進內質網壓力相關基因---------36、 結果討論-------------------------------38、 文獻參考--------------------------------41、 圖--------------------------------------45、 表--------------------------------------68錄-------------------------------------------70application/pdf9552502 bytesapplication/pdfen-US斑馬魚脂肪肝類胰島素生長因子肝癌zebrafishfatty liversteatosisIMP2HCCIGF2BP2[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/181646/1/ntu-96-R94b45025-1.pdf