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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉秉慧(Biing-Hui Liu) | |
| dc.contributor.author | Ya-Chih Cheng | en |
| dc.contributor.author | 鄭雅之 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:20:31Z | - |
| dc.date.available | 2022-09-08 | |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-21 | |
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H., . . . Fann, M. J. (2010). Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation. J Neurosci, 30(12), 4428-4439. doi:10.1523/JNEUROSCI.0473-10.2010 Xie, J., Farage, E., Sugimoto, M., & Anand-Apte, B. (2010). A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development. BMC Dev Biol, 10, 76. doi:10.1186/1471-213x-10-76 Yeh, C. W., & Hsu, L. S. (2016). Zebrafish diras1 Promoted Neurite Outgrowth in Neuro-2a Cells and Maintained Trigeminal Ganglion Neurons In Vivo via Rac1-Dependent Pathway. Mol Neurobiol, 53(10), 6594-6607. doi:10.1007/s12035-015-9550-2 Yu, M. W., Zhang, Y. J., Blaner, W. S., & Santella, R. M. (1994). Influence of vitamins A, C, and E and beta-carotene on aflatoxin B1 binding to DNA in woodchuck hepatocytes. Cancer, 73(3), 596-604. Yuan, T. F., Gu, S., Shan, C., Marchado, S., & Arias-Carrion, O. (2015). Oxidative Stress and Adult Neurogenesis. Stem Cell Rev, 11(5), 706-709. doi:10.1007/s12015-015-9603-y Yunus, A. W., Razzazi-Fazeli, E., & Bohm, J. (2011). Aflatoxin B(1) in affecting broiler's performance, immunity, and gastrointestinal tract: a review of history and contemporary issues. Toxins (Basel), 3(6), 566-590. doi:10.3390/toxins3060566 Zhang, C., Wang, J., Lu, G., Li, J., Lu, X., Mantion, G., . . . Lin, R. (2012). Hepatocyte proliferation/growth arrest balance in the liver of mice during E. multilocularis infection: a coordinated 3-stage course. PLoS One, 7(1), e30127. doi:10.1371/journal.pone.0030127 Zhang, W., & Dolan, M. E. (2010). The emerging role of microRNAs in drug responses. Curr Opin Mol Ther, 12(6), 695-702. Zorn, A. M., & Wells, J. M. (2009). Vertebrate endoderm development and organ formation. Annu Rev Cell Dev Biol, 25, 221-251. doi:10.1146/annurev.cellbio.042308.113344 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68421 | - |
| dc.description.abstract | 黃麴毒素B1（Aflatoxins B1, AFB1）為黃麴黴 (Aspergillus flavus) 等黴菌產生之二級代謝產物，已知其對人類及動物具有肝毒性及致肝癌性。目前對於AFB1在胚胎發育上的影響及毒性機制尚不明瞭，因此本研究將利用斑馬魚胚胎作為動物模式來探討AFB1對於胚胎發育的影響。實驗結果發現，AFB1處理斑馬魚胚胎達120 hpf (hours post fertilization) 後，雖然導致胚胎的高死亡率，卻不會對胚胎造成明顯的不良型態變化。以0.25及0.5 μM的AFB1處理轉殖肝螢光魚胚胎Tg（lfabp：eGFP）以觀察AFB1對肝臟發育的影響 (6-96 hpf)，結果顯示肝臟螢光強度隨毒素劑量上升而顯著下降，組織切片分析也顯示肝臟有縮小現象。末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記螢光染色 (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)的結果指出AFB1會促進肝臟細胞凋亡，磷酸化組蛋白H3免疫染色 (phospho-histone H3 immunostaining) 的結果則顯示AFB1可能可以抑制肝臟細胞增生。以prox1和hhex探針進行全胚體原位雜交法 (Whole-mount in situ hybridization) ，發現胚胎肝臟在特化 (specification) 階段（24hpf）就會受到AFB1的傷害，進一步在肝臟發育不同的不同時期以AFB1處理轉殖肝螢光魚胚胎Tg（lfabp：eGFP）觀察，得知肝臟在出芽/分化 (budding/ differentiation) 及增長 (outgrowth) 階段都有可能受AFB1影響而造成肝臟縮小的情形。AFB1的暴露亦會導致肝臟特異性的miR-122表現下降，但不會影響胚胎中凝血因子基因（f7、f3a、f3a）和肝臟特異性基因（vtna、cp）的表現。此外AFB1會誘發斑馬魚肝細胞（ZFL）中的氧化壓力上升，而抗氧化劑N-乙醯半胱氨酸（N-acetylcysteine , NAC）和維生素C (Vitamin C, Vit C) 的使用可以降低AFB1造成的細胞傷害、胚胎死亡率，而維生素C更可能可以改善肝臟縮小的情形。
過去研究指出AFB1也會造成中樞神經的損傷。我們由行為測試觀察到AFB1的暴露造成斑馬魚胚胎異常的游泳模式，同時也使胚胎移動的距離下降，因此推測AFB1可能會影響胚胎發育階段的神經生長。早期神經元的標記（HuC）和神經毒性的生物指標（gfap）的mRNA變化證實了AFB1會影響胚胎神經發育而產生神經毒性。乙醯化微管蛋白免疫染色 (Acetyl-alpha Tubulin staining, AcTub staining) 的結果也顯示，0.25及0.5 μM的AFB1明顯干擾了24 hpf胚胎神經元的發育。將 6 hpf 斑馬魚胚胎以 0.5 μM AFB1 處理至 48 hpf 後進行微陣列技術分析(microarray)，發現多個和神經發育相關基因的表現皆受到AFB1的影響。以半定量及定量聚合酶鏈鎖反應 (PCR) 確認在處理AFB1達24h及48h的胚胎中，ngfa 及atp1b1b mRNA的表現量顯著下降prtga mRNA的表現量則上升，上述結果表示AFB1可能可以干擾神經元的能量來源並抑制斑馬魚胚胎神經元的早期分化。 本篇研究證實AFB1會干擾早期胚胎肝臟和神經的發育，而相關的發育毒性的機制仍需要進一步的實驗來確認。 | zh_TW |
| dc.description.abstract | Aflatoxins B1 (AFB1) is a naturally occurring mycotoxin produced by the Aspergillus flavus group of fungi. It is also a well-known hepatocarcinogen that contributes significantly to the high incidence of hepatocellular carcinoma. Since there are few studies examining the developmental toxicity of AFB1, we used the zebrafish embryo as a vertebrate model herein. Treatment of zebrafish embryos from 6 hours post-fertilization (hpf) to 120 hpf resulted in a high mortality rate, but the morphology of embryos remained unchanged. To observe the effect of AFB1 on liver development, the embryos of transgenic zebrafish Tg(lfabp:eGFP) were treated with AFB1 ranging from 0.25 to 0.5 μM. We found that the liver fluorescence was decreased in a dose-dependent manner and histological analysis also indicated a shrunk liver size. TUNEL assay revealed that AFB1 promoted liver apoptosis and pH3 immunostaining implied that AFB1 might also inhibited liver proliferation. Whole-mount in situ hybridization with prox1 and hhex probes and observation of Tg(lfabp:eGFP) showed that the embryonic liver was affected by AFB1 at the specification and the budding/ differentiation stages (24-72hpf). Also, AFB1 treatment led to the down-regulation of liver-specific miR-122. However, AFB1 did not affect the gene expression of coagulation factors (f7, f3a, and f3a) and liver-specific genes (vtna and cp) in 6-72 hpf embryos. As AFB1 was found to increase the oxidative stress in zebrafish liver cell line (ZFL), ZFL cell death as well as embryo mortality mediated by AFB1 was restored in the presence of antioxidants N-acetylcysteine (NAC) and Vitamin C (Vit C). In addition, Vit C is likely to rescue the liver shrinkage that caused by AFB1.
Previous studies indicate that AFB1 also leads to brain damage. From our results of behavior test, AFB1 exposure decreased the total distance moved of embryos which swam in an abnormal shaking patterns. Therefore, we hypothesized that AFB1 might affect the neurodevelopment during developmental stage of embryos. The marker of early born neurons (HuC) and the biomarker of neurotoxicity (gfap) were both modulated by AFB1. Additionally, acetylated alpha-tubulin (AcTub) staining indicated that AFB1 disrupted the development of embryonic neurons. Microarray profiles of embryos after 48 hr AFB1 treatment showed an alteration of several genes which were related to neurodevelopment, including nerve growth factor a (ngfa), atp1b1b, and protogenin homolog a (prtga). PCR analysis further confirmed that AFB1 significantly decreased the expression of both ngfa and atp1b1b, and increased that of prtga gene in 6-48 hpf embryos, which suggest that AFB1 might have the ability to suppress neuronal differentiation and reduce the source of energy demand in neurons. In conclusion, AFB1 might interfere with the embryonic liver and neural development. Further experiments are required to confirm the mechanism of developmental hepatotoxicity and neurotoxicity associated with AFB1. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:20:31Z (GMT). No. of bitstreams: 1 ntu-106-R04447006-1.pdf: 2973527 bytes, checksum: fd1a056ef081c5ad12a5b5f36c36987d (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 摘要 III Abstract V Chapter 1. Introduction 1 1.1 Mycotoxin 1 1.2 Aflatoxin B1 2 1.2.1 Worldwide Occurrence and Limits of safe intake 4 1.2.2 Hepatotoxicity of AFB1 5 1.2.3 Neurotoxicity of AFB1 7 1.2.4 Developmental Toxicity of AFB1 8 1.2.5 Apoptosis and Oxidative Stress 9 1.3 Zebrafish (Danio rerio) 11 1.3.1 Zebrafish as a Model for Investigating Developmental Toxicity 11 1.3.2 Liver development of Zebrafish 14 1.3.3 Neural development of Zebrafish 15 1.4.1 Purpose of this study 17 1.4.2 The approach and finding in this study 18 Chapter 2. Methods and Materials 21 2.1 Materials 21 2.1.1 Experimental animals 21 2.1.2 Cell culture 21 2.1.3 Chemicals 22 2.1.4 Reagents 23 2.1.5 Instruments and Equipment 24 2.2 Methods 25 2.2.1 AFB1 exposure 25 2.2.2 Observation of liver, neural and vascular development 26 2.2.3 Paraffin sectioning 27 2.2.4 Hematoxylin and eosin stain, HE stain 27 2.2.5 Whole mount in-situ hybridization (WHISH) 28 2.2.6 TdT-mediated dUTP-biotin nick end labeling assay, TUNEL assay 29 2.2.7 Phosphohistone H3 (pH3) immunohistochemistry staining 30 2.2.8 Total RNA extraction 30 2.2.9 Reverse transcription reaction, RT reaction 31 2.2.10 Polymerase chain reaction, PCR 31 2.2.11 SYBR real-time polymerase chain reaction, RT-PCR 32 2.2.12 TaqMan real-time Polymerase Chain Reaction, RT-PCR 32 2.2.13 MTT assay 33 2.2.14 Cellular Reactive Oxygen Species Detection Assay (DCFH-DA) 34 2.2.15 Recording of zebrafish behavior 34 2.2.16 Microarray 35 2.2.17 Whole mount acetylated α-tubulin staining 36 2.2.18 Statistical analysis 36 Chapter 3. Results 38 3.1 Adverse effects of AFB1 on zebrafish embryos 38 3.2 AFB1 affected liver development in zebrafish embryos 38 3.3 AFB1 interfered with the liver at specification stage 39 3.4 AFB1 was likely to damage all three stages of liver development 40 3.5 AFB1 promoted hepatic cell apoptosis in embryonic zebrafish and might also inhibit the hepatic cell proliferation 40 3.6 AFB1 did not alter the expression of liver-specific genes and coagulation factor genes 41 3.7 AFB1 decreased the expression of liver-specific miR-122 42 3.8 AFB1 induced cytotoxicity and oxidative stress in ZFL 42 3.9 Antioxidants rescued the damage caused by AFB1 in ZFL 43 3.10 Antioxidants promoted the survival rate of AFB1-treated embryos 44 3.11 Antioxidants might rescue the AFB1-induced liver shrinkage 44 3.12 AFB1 contributed to the abnormal behavior of embryos in the early stage of development 45 3.13 AFB1 decreased the expression of early neuronal marker and induced embryonic neurotoxicity 45 3.14 AFB1 disrupted the embryonic neural development 46 3.15 AFB1 altered the expression of gene related to neurogenesis in zebrafish embryos 47 3.16 AFB1 showed a slight effect on brain vessel development 48 Chapter 4. Discussion 49 References 58 Tables 70 Figures 73 | |
| dc.language.iso | en | |
| dc.subject | 肝臟 | zh_TW |
| dc.subject | 神經 | zh_TW |
| dc.subject | 發育 | zh_TW |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | 黃麴毒素 | zh_TW |
| dc.subject | Aflatoxin B1 | en |
| dc.subject | zebrafish | en |
| dc.subject | neuron | en |
| dc.subject | liver | en |
| dc.subject | development | en |
| dc.title | 黃麴毒素B1對斑馬魚胚胎發育的影響 | zh_TW |
| dc.title | Effects of Aflatoxin B1 on the Development of Zebrafish Embryos | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許濤(Tao Hsu),陳曜鴻(Yau-Hung Chen) | |
| dc.subject.keyword | 黃麴毒素,斑馬魚,發育,肝臟,神經, | zh_TW |
| dc.subject.keyword | Aflatoxin B1,zebrafish,development,liver,neuron, | en |
| dc.relation.page | 96 | |
| dc.identifier.doi | 10.6342/NTU201704094 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-21 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 毒理學研究所 | zh_TW |
| 顯示於系所單位： | 毒理學研究所 | |
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