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研究生:李珮君
研究生(外文):Pei-chun Li
論文名稱:FGF10/FGFR2訊息影響斑馬魚肝臟之發育
論文名稱(外文):FGF10/FGFR2 signaling affects liver development of zebrafish
指導教授:王文柄
指導教授(外文):Wen-pin Wang
學位類別:碩士
校院名稱:慈濟大學
系所名稱:人類遺傳研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:71
中文關鍵詞:LFABP斑馬魚肝臟發育纖維母細胞生長因子
外文關鍵詞:LFABPFibroblast growth factor (FGF)liver developmentzebrafish
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纖維母細胞生長因子(FGF)訊息傳遞路徑曾被報導過在哺乳類動物的肝臟形成中扮演很重要的角色。我們篩選斑馬魚不同發育時期肝臟中,纖維母細胞生長因子接受器(FGFR)的表現情形,發現FGFR2是肝臟主要表現的亞型。我們又進一步找到FGFR2b的配體之一,FGF10,在早期肝臟發育中有較高的表現。然而,FGF10/FGFR2訊息傳遞路徑在斑馬魚肝臟發育所扮演的角色,仍是未知的。我們注射反股寡核苷酸(morpholino)到LFABP-GFP轉殖魚的胚胎中,以降低FGF10的表現量。此LFABP-GFP轉殖魚是以肝臟型脂肪酸結合蛋白(LFABP)的驅動子控制綠色螢光蛋白質表達,所以會在魚的肝臟有綠色螢光。結果顯示注射過FGF10 MO或FGFR2 MO的胚胎(morphant),其肝臟都比正常的還小,尤其是同時注射FGF10 MO和FGFR2 MO的double morphant。接著利用全覆式原位雜交法來偵測,發現肝臟專一表達的LFABP基因,在FGFR2 morphant 或double morphant中,其表達量是降低的。另外FGF下游基因pea3的表現在各種morphant中也減少,尤其是FGF10 morphant。我們將會把FGF10突變的斑馬魚,Daedalus (Dae),和LFABP-GFP轉殖魚做交配,以產生Dae/LFABP轉殖魚。然後直接於魚體中觀察其肝臟綠色螢光蛋白質表達情形,以及做各種組織學上的分析。另一方面,我們使用肝臟專一表達的LFABP驅動子異位地大量表達FGF10於肝臟中。我們將FGF10選殖到LF2.8-MCS-V5/CMV-RFP的雙功能載體中,並且將其注射到胚胎中。注射過因而含有紅色螢光蛋白質表達的胚胎將用於分析外源性基因的測定。在未來,我們會創造出在肝臟中穩定表達FGF10的轉殖魚,以應用在各種研究上。
The importance of fibroblast growth factor (FGF) signals in liver organogenesis has been reported in mammals. We screened the expression of FGF receptors (FGFR) in three developmental stages of zebrafish liver and found the FGFR2b was the major subtype. We also found FGF10, one of the ligands of the FGFR2b, expressed at high level in early liver development. However, the roles of FGF10/FGFR2 in liver development of zebrafish are not clear. We knocked down the expression of FGF10 by injecting antisense morpholino (MO) into the embryos of liver type fatty acid binding protein (LFABP)-GFP transgenic fish. The results indicated that the liver of FGF10 and FGFR2 morphants were smaller than wild-type, especially in FGF10/FGFR2 double morphants. Using whole-mount in situ hybridization, the expression pattern of liver specific gene LFABP was reduced in FGFR2 morphant and double morphant. The expression of downstream target gene pea3 was decreased in the morphants, especially in FGF10 morphants. The daedalus (dae) zebrafish, in which the FGF10 gene was disrupted, will crossed with the LF2.8-GFP transgenic fish to generate the dae/LFABP fish. The fish will be analyzed by direct observation of GFP expression in the liver and subsequent histological analysis. On the other hand, we used the liver-specific L-FABP promoter to ectopically overexpress FGF10 in the liver. We cloned FGF10 into the LF2.8-MCS-V5/CMV-RFP bi-functional vector and injected to embryos. The injected embryos with RFP will be analyzed for the expression of exogenous genes. In the future, we will create the stable transgenic fish lines with FGF10 overexpression.
1. Introduction 1
1.1. The development of mammalian liver 1
1.2. The development of zebrafish liver 4
1.3. The Fibroblast growth factor (FGF) signals in mammalian liver
development 7
1.4. Using zebrafish as a model to study liver development 10
1.5. The specific aims of this research 13
2. Materials and Methods 14
2.1. Materials 14
2.2. Methods 21
3. Results 33
3.1. Expression of FGF-related genes in three developmental stages of
zebrafish liver 33
3.2. Loss of function for FGFR2/FGF10 signaling 35
3.3. Gain of function for FGF10 signaling 39
4. Discussion 40
5. References 46
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