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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30201

Title: 藉由CB1R和ChREBP在斑馬魚肝臟中雙向的表現以建立早期脂肪肝動物模型
The Establishment of Early Hepatic Steatosic Models by Synergistic Expression of CB1R and ChREBP in Zebrafish Liver
Authors: Hsi-Wen Pang
Contributors: NTOU:Institute of Bioscience and Biotechnology
Keywords: 斑馬魚;脂肪肝
zebrafish;hepatic steatosis;CB1R;ChREBP
Date: 2011
Issue Date: 2011-11-25T07:36:22Z
Abstract: 近年來非酒精性脂肪肝已經成為患病率最高之慢性肝臟疾病,同時其也和肥胖、糖尿病以及高血壓等疾病有高度關聯性,因此非酒精性脂肪肝之研究越來越受到重視。高脂肪飲食以及飲酒會造成脂肪累積在肝臟,最主要的原因為脂質代謝方面出了問題。Carbohydrate response element-binding protein (ChREBP)是個存在於肝臟中的轉錄因子,此轉錄因子會因肝臟中葡萄糖濃度上升而促使其結合上Carbohydrate response element (ChoRE),並促使糖解反應酵素L-PK以及脂肪生成反應酵素ACC和FAS生成而導致脂肪的生成。另一方面,cannabinoid receptor type 1 (CB1R)也被認為和能量平衡以及脂肪製造有關係,當脂肪肝產生時,都可以發現CB1R量有明顯上升。因此我們設計出藉由L-FABP可以專一性在肝臟表現的可調控式複合載體,藉由此載體,當加入誘導物質時可讓基因被表現。接著利用此載體攜帶ChREBP和CB1R基因,並利用顯微注射法將此可調控式複合構築體打入野生型斑馬魚受精卵當中,篩選出第一代斑馬魚(F0)。藉由早期誘導第一代斑馬魚(F0)與野生型斑馬魚所生下的子代(F1),利用Oil Red O染色可以觀察到有一定程度的脂肪累積在肝臟。之後利用特別設計的可誘導式複合載體,當此載體被誘導之後,會自行產生轉錄因子而可以在短時間內大量表現欲表現之基因,利用此載體之特性,將其搭載上和脂肪生成相關基因ChREBP、CB1R以及PPARγ,並且將此可誘導式複合構築體藉由顯微注射法打入野生型斑馬魚受精卵內,同樣以早期誘導之方式,利用Nile Red脂肪螢光染色可以觀察到脂肪累積在肝臟之情況出現。因此可以確立藉由此兩種載體搭載調控脂肪生成之基因,可以於早期誘導而生成脂肪肝,而利於非酒精性脂肪肝之相關研究。
In recent years, non-alcoholic fatty liver disease (NAFLD) has become the highest prevalence of chronic liver disease, and its also been highly correlated with some other human chronic disease, e.g. obesity, diabetes or hypertension. High-fat diet and alcohol intake will produce fatty acid accumulation in liver, the main reason for fatty acid abnormal accumulate in the liver might be hepatic lipid metabolism dysfunction. Carbohydrate response element-binding protein (ChREBP) is a transcription factor, which is regulated by hepatic glucose concentration, will promote the rate-limiting enzyme of glycolysis, L-PK, and lipogenesis, ACC and FAS. On the other hand, cannabinoid receptor type 1 (CB1R) has been considered its relationship with energy homeostasis and fatty acid synthesis, which expression of CB1R will induce hepatic steatosis. According to this, we designed a regulatable complex mosaic vector system with L-FABP promoter, which can specifically expressed in liver. With inducing substances added, this vector system will subsequently make gene express. We use this verctor system to carry ChREBP and CB1R gene, therefore we inject this regulatable complex mosaic construct into the wild-type zebrafish zygotes. After that we screened the F0 zebrafish. Then we crossed the F0 zebrafish with the wild type zebrafish to obtain the offspring (F1). Finally, induced by Oil Red O stain of the 5dpf embryo for two days to observed the fat accumulation in the liver. We also designed another special inducible complex mosaic vector system, once this vector system been induced, it will produces its own transcription factors and overexpressed the interested genes in a short time. By the characteristics of this vector system, we carried lipogenesis-related genes, ChREBP, CB1R, and PPARγ on this vector system. We injected this inducible complex mosaic constructs into the wild-type zebrafish zygotes, then add the inducing substances in 5dpf stage for 2 days. By using Nile Red lipid fluorescent stain, we can observe the situation of fatty acids accumulated in liver. According to our research, we found that to regulate the gene of fatty acid synthesis by these two vector systems can induce steatosis in early stage, and may be useful in the research of non-alcoholic fatty liver disease.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98360028
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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