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標題: | 建立篩選改善非酒精性脂肪肝之機能性成分的雞隻模式 A Chicken Model for Functional Screening of Ingredients Ameliorating Non-alcoholic Fatty Liver |
作者: | Ting-Wei Huang 黃亭維 |
指導教授: | 丁詩同(Shih-Torng Ding) |
關鍵字: | 雞隻動物模式,非酒精性脂肪肝,膽固醇,膽鹼,機能性成分, chicken model,non-alcoholic fatty liver,functional ingredients,choline,cholesterol, |
出版年 : | 2021 |
學位: | 碩士 |
摘要: | 近年來,隨著肥胖盛行率升高,非酒精性脂肪肝病 (non-alcoholic fatty liver disease, NAFLD) 已是現代常見的慢性肝臟疾病之一。非酒精性脂肪肝病隨著病徵程度輕微從脂肪肝 (simple fatty liver)、非酒精性脂肪肝炎 (non-alcoholic steatohepatitis, NASH)、肝纖維化與硬化 (liver fibrosis and cirrhosis)、嚴重則會發展成肝癌 (hepatocellular carcinoma, HCC)。由於人類和雞隻生合成脂肪的主要器官皆為肝臟,因此本研究擬建立雞隻有效形成脂肪肝模式,以利後續篩選出能防治或改善脂肪肝的機能性成分。 本研究分為三部分,第一部分擬透過不同飼糧配方以快速誘導雞隻發生脂肪肝,實驗將七週齡伊莎蛋雞和蘆花雞各分成四組,分別餵飼以下飼糧配方,依序為控制組 (Control, 17% CP, 5.3% fat, and 1,300 mg/kg choline)、低蛋白高脂肪組 (LPHF, 13% CP, 9.1% fat, and 1,300 mg/kg choline)、高膽固醇低膽鹼組 (CLC, 17% CP, 7.6% fat with additional 2% cholesterol, and 800 mg/kg choline) 及低蛋白高脂肪高膽固醇低膽鹼組 (LPHFCLC, 13% CP, 12.6% fat with additional 2% cholesterol, and 800 mg/kg choline),每隔兩週會測量生長性狀及採血分析生理生化性狀和標記蛋白質濃度,實驗餵飼四週後犧牲採樣。結果發現,蘆花雞和伊莎蛋雞其高膽固醇低膽鹼組以及低蛋白高脂肪高膽固醇低膽鹼組的脂肪肝評分皆較高,組織切片有明顯油滴堆積,肝臟中三酸甘油酯和膽固醇濃度顯著上升,伊莎蛋雞血漿中測量到的生物標記dipeptidyl-peptidase 4 (DPP4) 也明顯升高,證實這兩組飼糧配方在肉雞和蛋雞上皆能夠成功誘導脂肪肝的發生。 第二部分實驗利用前述成功的高膽固醇低膽鹼 (CLC) 模式篩選能防治或改善脂肪肝的機能性成分,進行預防組和治療組實驗,預防組餵飼七週齡伊莎蛋雞CLC飼糧,並分別額外添加0.5%北蟲草、1%北蟲草、0.5%靈芝或1%靈芝,共五組,實驗為期四週犧牲;治療組分為兩部分,第一部分餵飼七週齡伊莎蛋雞CLC飼糧四週以誘導脂肪肝後,再依七週齡血脂濃度隨機分為三組餵飼Control、Control添加0.5%靈芝、Control添加1%靈芝,為期四週後犧牲採樣;治療組第二部分餵飼七週齡伊莎蛋雞CLC飼糧四週以誘導脂肪肝後,依七週齡血脂濃度隨機分為三組餵飼CLC、CLC添加0.5%靈芝、CLC添加1%靈芝,為期四週後犧牲採樣。結果顯示,預防組餵飼CLC以誘導脂肪肝發生的同時,在飼糧中添加0.5%或1%靈芝有降低雞隻脂肪肝的趨勢;治療組第一部分餵飼CLC成功誘發脂肪肝後,改以Control餵飼四週,組織切片中的油滴堆積程度顯著下降,能有效達到治療雞隻脂肪肝的效果;治療組第二部分在飼糧中添加不同濃度靈芝並無明顯抑制脂肪肝之效果。 先前的實驗結果證實透過餵飼四週CLC飼糧後,能成功誘導雞隻發生脂肪肝,而第三部分實驗預期建立快速且誘導脂肪肝程度低於CLC飼糧之模式,以利後續建立可篩選出有效減緩脂肪肝之機能性產物的平台,因此以七週齡伊莎蛋雞餵飼有無添加膽鹼 (+/-)、膽固醇濃度0%、0.5%、1%和2%,進行2x2複因子設計,實驗為期六週,結果顯示,膽固醇與膽鹼之間並無發生交互作用,餵飼1%和2%膽固醇的四組飼糧皆能誘導雞隻形成脂肪肝,而無添加膽鹼之組別,其誘導雞隻形成脂肪肝的程度顯著高於正常添加膽鹼之組別。 綜上所述,為了未來能利用此誘導年輕雞隻形成脂肪肝之模式作為能篩選出更準確的機能性產物以達到預防及治療脂肪肝效果的平台,建議使用正常添加膽鹼之組別,並添加1%膽固醇作為最適當能快速誘導年輕雞隻形成脂肪肝的模式,以利後續可利用在篩選出有助於防治非酒精性脂肪肝的機能性成分。 Fatty liver diseases, common metabolic diseases in chickens, can lead to a decrease in egg production and sudden death of chickens. To solve problems caused by the disease, reliable chicken models of fatty liver disease are required. The pathogenesis of fatty liver diseases in chickens is accompanied by an imbalance in lipid homeostasis, such as hepatic lipid accumulation, transportation and metabolism. Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in humans caused by excessive lipid accumulation in livers. According that liver is the primary organ for de novo lipogenesis in chickens and humans, whereas both liver and adipose tissue contribute to de novo lipogenesis in rodents. In this study, we generate rapid chicken models for fatty liver development, seven-week-old ISA female chickens were fed with a control diet (CON, 17% dietary protein, 5.3% fat, and 1300 mg/kg choline), a low protein and high fat diet (LPHF, 13% dietary protein, 9.1% fat, and 1300 mg/kg choline), a high cholesterol with low choline diet (CLC, 17% dietary protein, 7.6% fat with additional 2% cholesterol, and 800 mg/kg choline), a low protein, high fat, high cholesterol and low choline diet (LPHFCLC, 13% dietary protein, 12.6% fat with additional 2% cholesterol, and 800 mg/kg choline) for 4 weeks. Our results showed that the CLC or LPHFCLC diet induced hyperlipidemia. Histological examination and hepatic lipid analysis indicated that the CLC and LPHFCLC diet induced fatty liver steatosis. Plasma dipeptidyl-peptidase 4, a biomarker of fatty liver disease in laying hens, was increased in chickens with the CLC or LPHFCLC diet. Pronounced hepatic ballooning and immune cell infiltration were observed in the steatotic livers in accompany with elevated levels of IL1β and LITAF mRNAs, suggesting that the CLC and LPHFCLC diet also provoked steatohepatitis. These diets also induced hepatic steatosis in Plymouth Rock chickens. Thus, the CLC and LPHFCLC diet can be used to generate a model for fatty liver diseases in different strains of chickens. In ISA chickens fed with CLC diet, hepatic PPARγ, SREBP1c, and FASN mRNA levels were elevated, suggesting that a lipogenesis was enhanced by the CLC treatment. Our results also showed that treatment with a high cholesterol and low choline diet low protein, high fat, high cholesterol and low choline diet for four weeks induced fatty liver disease in chickens. These diets can be utilized to rapidly generate chicken models for fatty liver research. The next step used different concentrations of Cordyceps militaris or Ganoderma lucidum with CLC diet to examined if their effects preventing or ameliorating fatty liver in chickens. The present results showed that 0.5% or 1.0% Ganoderma lucidum supplementation with CLC alleviated fatty liver development by CLC diet in the prevention group. Regardless of Ganoderma lucidum supplementation the induced chicken fatty liver by CLC diet 4 weeks, week alleviated in the curing group. Different concentrations of Ganoderma lucidum failed to ameliorated fatty liver by CLC diet. Previous results confirmed that after four-week feeding with the CLC diets, chickens were successfully induced for fatty liver. So the last part of the experiment is to investigate the interaction dietary of high cholesterol and low choline levels to induce fatty liver. Besides, screening of functional components to prevent or alleviate fatty liver with assessed. Therefore, seven-week-old ISA laying hens were fed with a based diet containing high or low choline (+/-) and different levels of cholesterol (0%, 0.5%, 1% or 2%) in a 2x2 multiple factor design for six weeks. The result showed that the groups of diets fed with 1% and 2% cholesterol induced fatty liver in chickens, while the group with choline supplementation induced severe fatty liver than the normal choline group. Currently, most of the fatty liver models induced by diet in chickens are time consuming. To develop therapeutic methods for prevention of fatty liver diseases, a rapid and reliable chicken model of fatty liver diseases is required. Sum of all, since de novo lipogenesis is primary occurred in the liver both in chickens and human, 1% cholesterol with normal choline group is the best treatment to screen functional ingredients for preventing or ameliorating fatty liver in chickens. These diets can be utilized to rapidly generate chicken models for fatty liver research. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60294 |
DOI: | 10.6342/NTU202100473 |
全文授權: | 有償授權 |
顯示於系所單位: | 動物科學技術學系 |
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