核黃素對北京鴨生長發(fā)育和脂肪代謝的影響及其調控機制
發(fā)布時間：2018-09-11 19:43
【摘要】：本論文通過3個體內試驗研究了核黃素對北京鴨生長發(fā)育和脂肪代謝的影響及其調控機制,并通過體外試驗研究了核黃素對HepG2細胞增殖和線粒體功能的影響。試驗一旨在研究日糧核黃素水平對15-35日齡北京鴨生長發(fā)育的影響,并確定其需要量。本試驗設6個核黃素水平(1.38、2.38、3.38、4.38、5.38、6.38 mg/kg),選取288只體重相近的15日齡雄性北京鴨,隨機分為6個處理組,每個重復8只鴨。與飼喂基礎日糧(核黃素含量為1.38mg/kg)相比,添加核黃素可顯著提高平均日增重、平均日采食量、胸肌率、腹脂率、血漿及肝臟核黃素含量,顯著降低料重比和肝臟指數。以生產性能和組織核黃素為評價指標,采用折線模型估測15～35日齡北京鴨核黃素需要量為2.33～3.57mg/kg。試驗二旨在探究核黃素對生長前期北京鴨生長發(fā)育和脂肪代謝的影響及其調控機制。本試驗選取360只1日齡健康的雄性北京鴨,隨機分為三個處理組:核黃素缺乏組、采食配對組(人為控制該組與核黃素缺乏組采食量一致)和自由采食對照組,每個處理組12個重復,每個重復10只鴨,試驗期為21天。與采食配對組和自由采食對照組相比,核黃素缺乏顯著降低了生長前期北京鴨平均日增重,顯著提高了料重比和死亡率;顯著降低了血漿和肝臟核黃素含量;顯著提高了血漿和肝臟中甘油三酯和總膽固醇含量、肝臟總飽和脂肪酸、C6:0、C12:0、C16:0、C18:0含量和肝臟指數。這三組肝臟蛋白質組學分析顯示,與采食配對組和自由采食對照組相比,核黃素缺乏導致63個蛋白質表達量變化大于1.5倍,其中包括32個上調蛋白質和31個下調蛋白。GO聚類分析結果顯示差異蛋白主要富集在脂肪酸氧化和線粒體呼吸鏈電子傳遞過程。核黃素缺乏組中參與脂肪酸β氧化過程的ACADS、ACADM、ACAD9和ETFDH蛋白表達量下調,提示脂肪酸β氧化受損,脂肪分解減少,進而導致肝臟脂肪沉積。核黃素缺乏組中參與線粒體呼吸鏈電子傳遞過程的ACAD9、NDUFS1、NDUFA8和FXN蛋白表達量下調,提示呼吸鏈電子傳遞過程受損,進而導致ATP生成不足,影響動物生長。試驗三旨在研究種母鴨核黃素對母鴨繁殖性能和子代胚胎發(fā)育的影響。本試驗選取80只45周齡產蛋期北京鴨,隨機分為兩個組:核黃素缺乏組和對照組,分別飼喂核黃素添加量為0和10mg/kg的試驗日糧,試驗期為8周。種母鴨日糧核黃素對試驗期母鴨體重、產蛋率、蛋重、種蛋受精率和子代初生重沒有顯著影響;從試驗第2周開始,核黃素缺乏組種母鴨血漿核黃素和種蛋黃核黃素含量顯著降低,種蛋孵化率急劇降低,試驗第6周以后種蛋孵化率降為0。試驗第8周種蛋孵化第13天胚胎肝臟蛋白質組學分析結果顯示,種母鴨核黃素缺乏導致187個蛋白質表達量變化大于1.5倍,其中包括67個上調蛋白質和120個下調蛋白。KEGG通路分析顯示差異蛋白主要富集在三羧酸循環(huán)、脂肪酸β氧化和呼吸鏈電子傳遞等代謝過程。核黃素缺乏導致胚胎肝臟脂肪酸β氧化(ETFDH、CPT1A、ACSL1、ACADS、ACAT1、ACSL5、DECR1 和 ETFA)、三羧酸循環(huán)(DLD、SDHB、IDH1、SDHA 和 ACO1)和呼吸鏈電子傳遞(NDUFA9、NDUFS1、NDUFV1、NDUFA10和ACAD9)過程中蛋白表達下調,提示這些代謝過程可能受阻,進而導致ATP產生不足,導致胚胎發(fā)育不良甚至死亡。試驗四分為3個體外試驗,旨在研究核黃素對HepG2細胞增殖和線粒體功能的影響。試驗1研究了不同核黃素耗竭時間對HepG2線粒體功能的影響。HepG2細胞在核黃素缺乏和核黃素充足的培養(yǎng)基中分別培養(yǎng)2、4、6、8、10和12天,試驗結束時采用Seahorse測定細胞線粒體功能。與核黃素充足組相比,核黃素缺乏組HepG2細胞從試驗第2天起最大耗氧量和呼吸潛力顯著降低,隨著試驗周期的延長,最大耗氧量和呼吸潛力進一步降低,提示線粒體功能受損。試驗2研究了不同核黃素水平對HepG2細胞增殖和線粒體功能的影響。HepG2細胞在核黃素添加水平為0、0.5、5、10、20、40、100和1064nmol/L的培養(yǎng)基中培養(yǎng)8天。在試驗第8天,培養(yǎng)基中不添加核黃素組HepG2細胞數量、最大呼吸量和呼吸潛力顯著低于核黃素添加組,隨著核黃素添加水平的提高,這些指標逐漸提高,當核黃素水平分別提高到10、20和20nmol/L時到達平臺期。試驗3研究了補充核黃素對HepG2細胞增殖和線粒體功能的影響。HepG2細胞在不添加核黃素的培養(yǎng)基中培養(yǎng)8天,隨后在添加不同水平的核黃素(0、0.5、5和1064nmol/L)的培養(yǎng)基中培養(yǎng)4天。培養(yǎng)基中添加5nmol/L核黃素對試驗第4天細胞基礎呼吸量、最大呼吸量和呼吸潛力均有顯著的提高;培養(yǎng)基中添加1064nmol/L核黃素可顯著提高細胞數量,顯著提高基礎呼吸量、最大呼吸量和呼吸潛力,完全恢復線粒體呼吸功能。以上結果表明,HepG2細胞在不添加核黃素的培養(yǎng)基中培養(yǎng)2天后線粒體呼吸功能受損,8天后細胞增殖速率降低。培養(yǎng)基中添加20nmol/L核黃素可維持HepG2細胞細胞正常生長和線粒體呼吸功能。HepG2細胞在不添加核黃素的培養(yǎng)基中培養(yǎng)8天,培養(yǎng)基中添加1064nmol/L核黃素培養(yǎng)4天可顯著提高細胞增殖速率,并完全恢復線粒體呼吸功能。以上結果表明,核黃素缺乏可導致胚胎期和生長前期北京鴨生長發(fā)育不良、肝臟脂肪蓄積。肝臟蛋白質組學分析發(fā)現(xiàn),核黃素缺乏導致肝臟線粒體脂肪酸β氧化(ACADS、ACAD9和ETFDH)、呼吸鏈電子傳遞(ACAD9和NDUFS1)和三羧酸循環(huán)(DLD)過程中關鍵蛋白表達量下調,阻礙脂肪分解導致脂肪蓄積;阻礙能量生成,導致動物生長發(fā)育不良。體外試驗結果顯示,核黃素缺乏可導致HepG2細胞線粒體功能受損,補充核黃素后可恢復線粒體功能。
[Abstract]:The effects of riboflavin on the growth and fat metabolism of Beijing ducks were studied by three in vivo experiments. The effects of riboflavin on the proliferation and mitochondrial function of HepG2 cells were studied in vitro. The first experiment was designed to study the effects of dietary riboflavin levels on the growth and development of Beijing ducks aged 15-35 days. Six riboflavin levels (1.38, 2.38, 3.38, 4.38, 5.38, 6.38 mg/kg) were used to select 288 15-day-old Beijing ducks with similar body weight and randomly divided into six treatment groups with 8 ducks in each repetition. The riboflavin requirement of Beijing ducks aged 15-35 days was estimated by a broken-line model with the production performance and tissue riboflavin as the evaluation indexes. Experiment 2 was designed to explore the effects of riboflavin on the growth and lipid content of pre-growth Beijing ducks. In this study, 360 1-day-old healthy male Peking ducks were randomly divided into three groups: riboflavin deficiency group, feeding matching group (the same amount of food as riboflavin deficiency group) and free-feeding control group. Each group had 12 replicates, 10 ducks per replicate for 21 days. Riboflavin deficiency significantly reduced the average daily gain, increased the feed-to-weight ratio and mortality, decreased the riboflavin content in plasma and liver, increased the triglycerides and total cholesterol content in plasma and liver, and the total saturated fatty acids in liver, C6:0, C12, respectively. The three groups of liver proteomics analysis showed that the deficiency of riboflavin resulted in 63 protein expression changes more than 1.5 times, including 32 up-regulated proteins and 31 down-regulated proteins. The expression of ACADS, ACADM, ACAD9 and ETFDH proteins involved in the oxidation of fatty acid beta in the riboflavin deficient group was down-regulated, suggesting that fatty acid beta oxidation was impaired and fat decomposition was reduced, leading to liver fat deposition. The down-regulation of ACAD9, NDUFS1, NDUFA8 and FXN proteins suggests that the electron transport process in the respiratory chain is impaired, which leads to insufficient ATP production and affects animal growth. Riboflavin deficiency group and control group were fed with riboflavin supplement of 0 and 10 mg/kg for 8 weeks, respectively. Riboflavin had no significant effect on body weight, egg laying rate, egg weight, fertilization rate of breeding eggs and birth weight of offspring. From the second week of the experiment, plasma riboflavin and species of breeding ducks in riboflavin deficiency group were fed with riboflavin supplement of 0 and 10 mg/kg respectively. The content of riboflavin in egg yolk decreased significantly and the hatchability of eggs decreased sharply. The hatchability of eggs dropped to 0 after the 6th week of the experiment. Proteomic analysis of the liver of the embryos on the 13th day of the 8th week of the experiment showed that the riboflavin deficiency caused 187 protein expression changes more than 1.5 times, including 67 up-regulated proteins and 120 up-regulated proteins. KEGG pathway analysis showed that the differentially expressed proteins were mainly enriched in the tricarboxylic acid cycle, fatty acid beta oxidation and electron transport of respiratory chain. Riboflavin deficiency led to fatty acid beta oxidation (ETFDH, CPT1A, ACSL1, ACADS, ACAT1, ACSL5, DECR1 and ETFA), tricarboxylic acid cycle (DLD, SDHB, IDH1, SDHA and ACO1), and respiratory chain electricity in embryonic liver. The down-regulation of protein expression during Subtransmission (NDUFA9, NDUFS1, NDUFV1, NDUFA10, and ACAD9) suggests that these metabolic processes may be blocked, resulting in ATP deficiency, resulting in embryonic dysplasia and even death. Three in vitro trials were conducted to investigate the effects of riboflavin on the proliferation and mitochondrial function of HepG2 cells. HepG2 cells were cultured in riboflavin-deficient and riboflavin-rich medium for 2,4,6,8,10 and 12 days, respectively. Mitochondrial function was measured by Seahorse at the end of the experiment. Compared with riboflavin-deficient group, HepG2 cells in riboflavin-deficient group had the maximum oxygen consumption from the second day of the experiment. The effects of riboflavin levels on the proliferation and mitochondrial function of HepG2 cells were studied in experiment 2. HepG2 cells were cultured in medium with riboflavin levels of 0,0.5,5,10,20,40,100 and 1064 nmol/L. On the 8th day, the number of HepG2 cells in the medium without riboflavin was significantly lower than that in the riboflavin supplementation group. With the increase of riboflavin supplementation, these indexes gradually increased, reaching the plateau when the riboflavin levels increased to 10, 20 and 20 nmol/L, respectively. Effects of lutein on proliferation and mitochondrial function of HepG2 cells were studied. HepG2 cells were cultured for 8 days without riboflavin, and then for 4 days in medium with different levels of riboflavin (0,0.5,5 and 1064 nmol/L). The basal respiration, maximum respiration and respiratory potential of HepG2 cells were measured by adding 5 nmol/L riboflavin to the medium on day 4. The results showed that the mitochondrial respiratory function of HepG2 cells was impaired after 2 days of culture without riboflavin and 8 days after culture. The normal growth and mitochondrial respiratory function of HepG2 cells were maintained by adding 20 nmol/L riboflavin to the medium. HepG2 cells were cultured in the medium without riboflavin for 8 days and 1064 nmol/L riboflavin for 4 days. These results suggest that riboflavin deficiency can lead to maldevelopment and liver fat accumulation in Beijing ducks during embryonic and early growth stages. Proteomic analysis of liver revealed that riboflavin deficiency is the key factor in the process of fatty acid beta oxidation (ACADS, ACAD9 and ETFDH), respiratory chain electron transport (ACAD9 and NDUFS1) and tricarboxylic acid cycle (DLD) in liver mitochondria. The results of in vitro experiments showed that riboflavin deficiency could lead to mitochondrial dysfunction in HepG2 cells, and mitochondrial function could be restored after riboflavin supplementation.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S834.5
本文編號：2237683
[Abstract]:The effects of riboflavin on the growth and fat metabolism of Beijing ducks were studied by three in vivo experiments. The effects of riboflavin on the proliferation and mitochondrial function of HepG2 cells were studied in vitro. The first experiment was designed to study the effects of dietary riboflavin levels on the growth and development of Beijing ducks aged 15-35 days. Six riboflavin levels (1.38, 2.38, 3.38, 4.38, 5.38, 6.38 mg/kg) were used to select 288 15-day-old Beijing ducks with similar body weight and randomly divided into six treatment groups with 8 ducks in each repetition. The riboflavin requirement of Beijing ducks aged 15-35 days was estimated by a broken-line model with the production performance and tissue riboflavin as the evaluation indexes. Experiment 2 was designed to explore the effects of riboflavin on the growth and lipid content of pre-growth Beijing ducks. In this study, 360 1-day-old healthy male Peking ducks were randomly divided into three groups: riboflavin deficiency group, feeding matching group (the same amount of food as riboflavin deficiency group) and free-feeding control group. Each group had 12 replicates, 10 ducks per replicate for 21 days. Riboflavin deficiency significantly reduced the average daily gain, increased the feed-to-weight ratio and mortality, decreased the riboflavin content in plasma and liver, increased the triglycerides and total cholesterol content in plasma and liver, and the total saturated fatty acids in liver, C6:0, C12, respectively. The three groups of liver proteomics analysis showed that the deficiency of riboflavin resulted in 63 protein expression changes more than 1.5 times, including 32 up-regulated proteins and 31 down-regulated proteins. The expression of ACADS, ACADM, ACAD9 and ETFDH proteins involved in the oxidation of fatty acid beta in the riboflavin deficient group was down-regulated, suggesting that fatty acid beta oxidation was impaired and fat decomposition was reduced, leading to liver fat deposition. The down-regulation of ACAD9, NDUFS1, NDUFA8 and FXN proteins suggests that the electron transport process in the respiratory chain is impaired, which leads to insufficient ATP production and affects animal growth. Riboflavin deficiency group and control group were fed with riboflavin supplement of 0 and 10 mg/kg for 8 weeks, respectively. Riboflavin had no significant effect on body weight, egg laying rate, egg weight, fertilization rate of breeding eggs and birth weight of offspring. From the second week of the experiment, plasma riboflavin and species of breeding ducks in riboflavin deficiency group were fed with riboflavin supplement of 0 and 10 mg/kg respectively. The content of riboflavin in egg yolk decreased significantly and the hatchability of eggs decreased sharply. The hatchability of eggs dropped to 0 after the 6th week of the experiment. Proteomic analysis of the liver of the embryos on the 13th day of the 8th week of the experiment showed that the riboflavin deficiency caused 187 protein expression changes more than 1.5 times, including 67 up-regulated proteins and 120 up-regulated proteins. KEGG pathway analysis showed that the differentially expressed proteins were mainly enriched in the tricarboxylic acid cycle, fatty acid beta oxidation and electron transport of respiratory chain. Riboflavin deficiency led to fatty acid beta oxidation (ETFDH, CPT1A, ACSL1, ACADS, ACAT1, ACSL5, DECR1 and ETFA), tricarboxylic acid cycle (DLD, SDHB, IDH1, SDHA and ACO1), and respiratory chain electricity in embryonic liver. The down-regulation of protein expression during Subtransmission (NDUFA9, NDUFS1, NDUFV1, NDUFA10, and ACAD9) suggests that these metabolic processes may be blocked, resulting in ATP deficiency, resulting in embryonic dysplasia and even death. Three in vitro trials were conducted to investigate the effects of riboflavin on the proliferation and mitochondrial function of HepG2 cells. HepG2 cells were cultured in riboflavin-deficient and riboflavin-rich medium for 2,4,6,8,10 and 12 days, respectively. Mitochondrial function was measured by Seahorse at the end of the experiment. Compared with riboflavin-deficient group, HepG2 cells in riboflavin-deficient group had the maximum oxygen consumption from the second day of the experiment. The effects of riboflavin levels on the proliferation and mitochondrial function of HepG2 cells were studied in experiment 2. HepG2 cells were cultured in medium with riboflavin levels of 0,0.5,5,10,20,40,100 and 1064 nmol/L. On the 8th day, the number of HepG2 cells in the medium without riboflavin was significantly lower than that in the riboflavin supplementation group. With the increase of riboflavin supplementation, these indexes gradually increased, reaching the plateau when the riboflavin levels increased to 10, 20 and 20 nmol/L, respectively. Effects of lutein on proliferation and mitochondrial function of HepG2 cells were studied. HepG2 cells were cultured for 8 days without riboflavin, and then for 4 days in medium with different levels of riboflavin (0,0.5,5 and 1064 nmol/L). The basal respiration, maximum respiration and respiratory potential of HepG2 cells were measured by adding 5 nmol/L riboflavin to the medium on day 4. The results showed that the mitochondrial respiratory function of HepG2 cells was impaired after 2 days of culture without riboflavin and 8 days after culture. The normal growth and mitochondrial respiratory function of HepG2 cells were maintained by adding 20 nmol/L riboflavin to the medium. HepG2 cells were cultured in the medium without riboflavin for 8 days and 1064 nmol/L riboflavin for 4 days. These results suggest that riboflavin deficiency can lead to maldevelopment and liver fat accumulation in Beijing ducks during embryonic and early growth stages. Proteomic analysis of liver revealed that riboflavin deficiency is the key factor in the process of fatty acid beta oxidation (ACADS, ACAD9 and ETFDH), respiratory chain electron transport (ACAD9 and NDUFS1) and tricarboxylic acid cycle (DLD) in liver mitochondria. The results of in vitro experiments showed that riboflavin deficiency could lead to mitochondrial dysfunction in HepG2 cells, and mitochondrial function could be restored after riboflavin supplementation.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S834.5
【參考文獻】
相關期刊論文 前10條
1 辛中豪;高蔚娜;蒲玲玲;姚站馨;王亞雯;郭長江;;核黃素對HepG2細胞抗氧化功能的影響[J];營養(yǎng)學報;2016年04期
2 唐靜;謝明;聞治國;馮宇隆;黃葦;侯水生;;核黃素對北京鴨生長性能和抗氧化機能的影響[J];動物營養(yǎng)學報;2013年12期
3 唐靜;謝明;侯水生;黃葦;聞治國;朱勇文;;日糧核黃素水平對1～21日齡北京鴨生長性能、抗氧化能力及激素分泌的影響[J];畜牧獸醫(yī)學報;2012年11期
4 達瑞;劉永琦;;差異蛋白組學分析與鑒定方法的研究進展[J];吉林醫(yī)學;2010年33期
5 王艷輝;王安;謝富;;維生素B_2對籠養(yǎng)蛋雛鴨生長性能、內分泌及抗氧化能力的影響[J];動物營養(yǎng)學報;2009年01期
6 王志躍,汪張貴,龔道清,范剛,盛東峰,趙秀花;日糧核黃素水平對新?lián)P州仔雞免疫器官發(fā)育及體液免疫的影響[J];動物營養(yǎng)學報;2005年03期
7 張建海,劉玲,王俊東,郝俊虎,萬雙秀;核黃素對肉仔雞免疫功能和生產性能的影響[J];飼料研究;2003年03期
8 何大澄,肖雪媛;差異蛋白質組學及其應用[J];北京師范大學學報(自然科學版);2002年04期
9 梁惠芳,柳啟沛,徐京;核黃素對大鼠脂質過氧化影響的研究[J];衛(wèi)生研究;1999年06期
10 王雯慧,高齊瑜;雛雞核黃素缺乏癥的病理學研究[J];畜牧獸醫(yī)學報;1999年05期
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