一部分: 
早產兒視網膜病變是造成嬰兒眼睛失明主要原因之一，因局部缺氧所引發過度的血管新生，當血管侵犯到玻璃體時，會導致滲漏、出血、視網膜剝離等症狀。到目前為止，其治癒後情形改善仍有限，其中主要原因在於缺乏合適的疾病動物模式來加以研究和探討。本研究將血管發綠螢光基因轉殖斑馬魚 (fli1-EGFP) 浸泡於 CoCl2 中，用來模擬缺氧的環境。結果發現到斑馬魚視網膜血管有明顯異常增生的情形，接著我們注射特殊染劑進斑馬魚胚胎，發現到染劑因為這些不正常增生之血管而有滲漏的狀況。從 Q-RT-PCR 發現到vegfaa和vegfr2 表現上升至 2.00 及 3.74 倍，顯示利用化學方式模擬缺氧確實可以引發 VEGF 訊號的傳遞，進而形成不正常血管新生。於是我們利用 SU5416、bevacizumab 和 ranibizumab 已知可抗血管新生之藥物發現，因缺氧所造成的過渡生長的血管可以有效被抑制，甚至其染料滲漏的情形有顯著的改善。結果得知利用斑馬魚胚胎建立早產兒視網膜病變擁有很好的優勢，與臨床上所觀察得現象相當一致，同時這樣的動物疾病模式未來也可用於藥物篩選及開發。

第二部分: 
NF-Y 是 CCAAT 主要的結合轉錄因子，由 NF-YA、NF-YB、NF-YC 三種次單元所組成。本研究利用斑馬魚作為模式物種，來探討 nfyc 是否會影響眼睛的發育。nfyc 在斑馬魚存在有兩種型態：nfyc-tv1 (336 個胺基酸) 及 nfyc-tv2  (360 個胺基酸)；其中又以nfyc-tv1與其他物種序列最相似。接著顯微注射反股寡核苷酸 (morpholino, MO) 抑制內生性 nfyc 的蛋白轉譯後，發現抑制 nfyc 會造成斑馬魚眼睛明顯縮小且比例會隨著注射濃度增加而上升。注射pax6a MO 之斑馬魚外觀與注射nfyc MO 相似，另外進行 pax6a 抗體染色，發現抑制 nfyc 會影響其視網膜神經節細胞分化。進一步以 pax6a 的探針進行原位雜合反應，發現抑制 nfyc 後的早期表現無明顯改變，晚期訊號看似些微下降或許是太多死細胞造成。至於利用西方點墨法發現抑制nfyc會造成 pax6a蛋白質表現上升。而抑制 nfyc 後進行BrdU 染色及 TUNEL assay，結果顯示抑制 nfyc 會導致眼睛細胞增生數量減少，且在腦部及眼睛有細胞凋亡的現象。綜合以上實驗結果，推測 nfyc 可能透過影響斑馬魚視神經的發育，進而對視網膜的分化造成影響。

Part I:
Retinopathy of prematurity (ROP), formerly known as retrolental fibroplasia, is a leading cause of infantile blindness worldwide. In this disease, the failure of central retinal vessels reaching to the retinal periphery creates a non-perfused peripheral retina that results in retinal hypoxia, neovascularization, haemorrhage, fibrosis and loss of vision. We established a ROP model using a green fluorescent vascular endothelium zebrafish transgenic line (fli-EGFP) treated with cobalt chloride (CoCl2, a hypoxia-inducing agent) and followed by GS4012 (a vascular endothelial growth factor inducer) from 24 hpf, and we found that numbers of vascular branches and sproutings were significantly increased in the central retinal vascular trunks 3-5 days after treatment. We also created an angiography method using tetramethyl rhodamine-dextran which displayed severe vascular leakage through the vessel wall into the surrounding retinal tissue. Furthermore, real-time quantitative PCR revealed expression of vegfaa and vegfr2 to increase by 2.00 and 3.74 folds in comparison with the corresponding control group, indicating increased VEGF signalling in hypoxic condition. Our model showed a rapid growth of neovascularization from the retinal vessels that resemble the clinical features of ROP. Specifically, according to the effect of SU5416 bevacizumab and ranibizumab, we demonstrated that hypoxia-induced angiogenesis in the retina also requires the effects of VEGF. Our findings also provide a simple and highly reproducible, clinically relevant ROP model using zebrafish embryos, which may be served as a platform for understanding the mechanisms of ROP development and progression, and provide an efficient way to screen candidate drugs in the future.

Part II:
Nuclear factor-Y (NF-Y) is a CCAAT-box-binding transcription factor which is composed of three subunits (NF-YA, NF-YB, and NF-YC). In this study, we used zebrafish as an animal model to study their roles during early developmental stage. While endogenous nfyc was knocked down by antisense morpholino of nfyc (nfyc-MO), a reduction in eye size was observed in nfyc-MO-injected embryos compared with wild-type embryos. Immunostaining with neuron-specific antibodies (Zn8 and Pax6a) revealed that nfyc-MO affected nfyc expression in ganglion cell layer, suggesting that nfyc is associated with the development of retinal neurons. Based on immunostaining with BrdU, a decrease in proliferating cells was found in eyes of nfyc-MO-injected embryos. TUNEL assay results revealed the apoptosis in head and eyes of nfyc-MO-injected embryos. Our in situ hybridization data didn’t showed significant differences in pax6a pattern, but protein level demonstrated that reverse correlation between NFYC and Pax6a. Taken together, our results suggested that zebrafish nfyc may affect the development of retinal neurons, and further affect zebrafish eye development.

Abstract (Chinese) Ⅰ
Abstract (English) Ⅲ
Content	Ⅵ
Chart list Ⅶ

Part I
Introduction 2
Materials 8
Methods	9
Results	15
Discussion 21
Part II
Introduction 27
Materials 37
Methods	37
Results	45
Discussion 51
Reference 81

論文名稱：第一部分:利用斑馬魚研究早產兒視網膜病變
        第二部分:細胞核因子C次單元對斑馬魚眼睛之重要性

第二部分:細胞核因子C次單元對斑馬魚眼睛之
5 發育之重要性
	頁數：108
校系(所)組別：淡江大學	    化學	研究所
畢業時間及提要別：  101  學年度第  2  學期   碩士	學位論文提要
研究生：張櫂杬	指導教授：陳曜鴻 博士	
論文提要內容：  





第一部分: 
早產兒視網膜病變是造成嬰兒眼睛失明主要原因之一，因局部缺氧所引發過度的血管新生，當血管侵犯到玻璃體時，會導致滲漏、出血、視網膜剝離等症狀。到目前為止，其治癒後情形改善仍有限，其中主要原因在於缺乏合適的疾病動物模式來加以研究和探討。本研究將血管發綠螢光基因轉殖斑馬魚 (fli1-EGFP) 浸泡於 CoCl2 中，用來模擬缺氧的環境。結果發現到斑馬魚視網膜血管有明顯異常增生的情形，接著我們注射特殊染劑進斑馬魚胚胎，發現到染劑因為這些不正常增生之血管而有滲漏的狀況。從 Q-RT-PCR 發現到vegfaa和vegfr2 表現上升至 2.00 及 3.74 倍，顯示利用化學方式模擬缺氧確實可以引發 VEGF 訊號的傳遞，進而形成不正常血管新生。於是我們利用 SU5416、bevacizumab 和 ranibizumab 已知可抗血管新生之藥物發現，因缺氧所造成的過渡生長的血管可以有效被抑制，甚至其染料滲漏的情形有顯著的改善。結果得知利用斑馬魚胚胎建立早產兒視網膜病變擁有很好的優勢，與臨床上所觀察得現象相當一致，同時這樣的動物疾病模式未來也可用於藥物篩選及開發。

第二部分: 
NF-Y 是 CCAAT 主要的結合轉錄因子，由 NF-YA、NF-YB、NF-YC 三種次單元所組成。本研究利用斑馬魚作為模式物種，來探討 nfyc 是否會影響眼睛的發育。nfyc 在斑馬魚存在有兩種型態：nfyc-tv1 (336 個胺基酸) 及 nfyc-tv2  (360 個胺基酸)；其中又以nfyc-tv1與其他物種序列最相似。接著顯微注射反股寡核苷酸 (morpholino, MO) 抑制內生性 nfyc 的蛋白轉譯後，發現抑制 nfyc 會造成斑馬魚眼睛明顯縮小且比例會隨著注射濃度增加而上升。注射pax6a MO 之斑馬魚外觀與注射nfyc MO 相似，另外進行 pax6a 抗體染色，發現抑制 nfyc 會影響其視網膜神經節細胞分化。進一步以 pax6a 的探針進行原位雜合反應，發現抑制 nfyc 後的早期表現無明顯改變，晚期訊號看似些微下降或許是太多死細胞造成。至於利用西方點墨法發現抑制nfyc會造成 pax6a蛋白質表現上升。而抑制 nfyc 後進行BrdU 染色及 TUNEL assay，結果顯示抑制 nfyc 會導致眼睛細胞增生數量減少，且在腦部及眼睛有細胞凋亡的現象。綜合以上實驗結果，推測 nfyc 可能透過影響斑馬魚視神經的發育，進而對視網膜的分化造成影響。

關鍵字：斑馬魚、早產兒視網膜病變、缺氧、nfyc、眼睛







表單編號：ATRX-Q03-001-FM030-01
Title of Thesis:                                 Total pages:108
Part Ι: Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: an Efficient Model of Retinopathy of Prematurity
Part II: Importance of Zebrafish CCAAT Box Binding Transcription Factor (NF-Y) C Subunit During Eye Development

Key word:
zebrafish, Retinopathy of Prematurity, hypoxia, NF-YC , eye 

Name of Institute: 
Graduate Institute of Chemistry, Tamkang University 

Graduate date: June, 2013            Degree conferred: M.S.

Name of student:   Chang, Chao-Yuan    Advisor:  Chen, Yau-Hung
                  張櫂杬                        陳曜鴻     
Abstract:
Part I:
Retinopathy of prematurity (ROP), formerly known as retrolental fibroplasia, is a leading cause of infantile blindness worldwide. In this disease, the failure of central retinal vessels reaching to the retinal periphery creates a non-perfused peripheral retina that results in retinal hypoxia, neovascularization, haemorrhage, fibrosis and loss of vision. We established a ROP model using a green fluorescent vascular endothelium zebrafish transgenic line (fli-EGFP) treated with cobalt chloride (CoCl2, a hypoxia-inducing agent) and followed by GS4012 (a vascular endothelial growth factor inducer) from 24 hpf, and we found that numbers of vascular branches and sproutings were significantly increased in the central retinal vascular trunks 3-5 days after treatment. We also created an angiography method using tetramethyl rhodamine-dextran which displayed severe vascular leakage through the vessel wall into the surrounding retinal tissue. Furthermore, real-time quantitative PCR revealed expression of vegfaa and vegfr2 to increase by 2.00 and 3.74 folds in comparison with the corresponding control group, indicating increased VEGF signalling in hypoxic condition. Our model showed a rapid growth of neovascularization from the retinal vessels that resemble the clinical features of ROP. Specifically, according to the effect of SU5416 bevacizumab and ranibizumab, we demonstrated that hypoxia-induced angiogenesis in the retina also requires the effects of VEGF. Our findings also provide a simple and highly reproducible, clinically relevant ROP model using zebrafish embryos, which may be served as a platform for understanding the mechanisms of ROP development and progression, and provide an efficient way to screen candidate drugs in the future.

Part II:
Nuclear factor-Y (NF-Y) is a CCAAT-box-binding transcription factor which is composed of three subunits (NF-YA, NF-YB, and NF-YC). In this study, we used zebrafish as an animal model to study their roles during early developmental stage. While endogenous nfyc was knocked down by antisense morpholino of nfyc (nfyc-MO), a reduction in eye size was observed in nfyc-MO-injected embryos compared with wild-type embryos. Immunostaining with neuron-specific antibodies (Zn8 and Pax6a) revealed that nfyc-MO affected nfyc expression in ganglion cell layer, suggesting that nfyc is associated with the development of retinal neurons. Based on immunostaining with BrdU, a decrease in proliferating cells was found in eyes of nfyc-MO-injected embryos. TUNEL assay results revealed the apoptosis in head and eyes of nfyc-MO-injected embryos. Our in situ hybridization data didn’t showed significant differences in pax6a pattern, but protein level demonstrated that reverse correlation between NFYC and Pax6a. Taken together, our results suggested that zebrafish nfyc may affect the development of retinal neurons, and further affect zebrafish eye development.
























表單編號：ATRX-Q03-001-FM031-01
Content
Abstract (Chinese)	Ⅰ
Abstract (English)	Ⅲ
Content	Ⅵ
Chart list	Ⅶ

Part I
Introduction	2
Materials	8
Methods	9
Results	15
Discussion	21
Part II
Introduction	27
Materials	37
Methods	37
Results	45
Discussion	51
Reference	81



Chart list
Fig. 1. Comparative five days post-fertilization dose-survival curves of zebrafish	55
Fig. 2 CoCl2 induced ectopic SIV in both Tg(fli1:eGFP) and WT zebrafish embryos.	56
Fig. 3 CoCl2 treated zebrafish show an increased vegfaa and vegfr2 mRNA expression.	57
Fig. 4 Confocal analysis of mock Tg(fli1a:efgp) zebrafish taken at 40x objective with LSM 780	58
Fig. 5 Confocal comparison of control wildtype with CoCl2 and GS4012 with a two day time-lapse	59
Fig. 6 Fluorescent dye leakage analysis of 3dpf wildtype and treated zebrafish using 10,000MW Dextran.	60
Fig. 7 Fluorescent dye leakage analysis of 5dpf wildtype and treated zebrafish using 10,000MW Dextran	61
Fig. 8 Fluorescent dye leakage analysis of 5dpf wildtype and treated zebrafish using 2,000,000MW tetramethylrhodamine.	62
Fig. 9 Confocal analysis of zebrafish treated with 5mM CoCl2 and 0.5μM SU5416	63
Fig. 10 Fluorescent TAMRA dye injections of 10mM CoCl2 and 0.5μM SU5416 co-treated zebrafish at 3dpf (left) and 5dpf (right).	64
Fig. 11 Fluorescent TAMRA dye injections in different condition	65
Fig. 12 Angiogenesis and the Role of CoCl2 and VEGF	66
Fig. 13 Alignment of NF-YC amino acid sequences of 6 organisms	67
Fig. 14 Phylogenetic tree of evolutionary relationship based on alignments of NF-YC	68
Fig. 15 Sequence of ATG MO and the levels of phenotypes.	69
Fig. 16 Both NF-YC and Pax6a have each transcription binding site on the promoter region.	70
Fig. 17 Knocking down nf-yc and pax6a showed similar phenotypes.	71
Fig. 18 Antibody staining of both NF-YC and Zn8 in WT embryo	72
Fig. 19 Antibody staining of both Pax6a and Zn8 in WT embryo	73
Fig. 20 Antibody staining of both NF-YC and Zn8 in WT embryo and nf-yc-morphants.	74
Fig. 21 Antibody staining of both Pax6a and Zn8 in WT embyo and nf-yc-morphants	75
Fig. 22 mRNA expression of pax6a in WT embryos and nf-yc-morphants.	76
Fig. 23 mRNA expression of nf-yc in pax6a-morphants showed significant increased.	77
Fig. 24 mRNA expression of six3b in nf-yc-morphants didn’t show significant changes.	78
Fig. 25 Western blotting analysis of the protein extracted from WT embryos and nf-yc-morphants using antibody against Pax6a protein	79
Fig. 26 Knocking NF-YC down results in cell apoptosis in the eye	80
Fig. 27 Knocking NF-YC down results in cell proliferation being inhibited	81



Table 1 qPCR primer sequences	82
Table 2 Amino acid sequences similarity (%)	83
Table 3 Dose response of phenotypes caused by nf-yc-MO injections	84
Table 4 Dose response of different degree of phenotypes caused by nf-yc-MO injections	85
Table 5 Dose response of phenotypes resued co-injection of nf-yc-MO and capped zebrafish nf-yc mRNA	86

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