应激对肉仔鸡小肽吸收的影响及小肽调控脂肪代谢的机理研究
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摘要
本论文以肌苷肽(Gly-Sar)及酪啡肽(β-CM-3)为试验材料,分别研究肉仔鸡小肽吸收过程中应激的影响机制以及小肽吸收后对肉仔鸡脂肪代谢的调控。
试验一旨在研究糖皮质激素类物质地塞米松对肉仔鸡生产性能及血浆内源皮质酮含量的影响。试验选取体重相近的21日龄雄性AA肉仔鸡200只,随机分为4个处理,每个处理5个重复,每个重复10只鸡。其中3个处理组腹部皮下注射地塞米松,注射剂量分别为0.1、0.5、2.5mg/kgBW,对照组注射等体积生理盐水,连续注射7天。结果表明,与对照组相比地塞米松显著降低肉仔鸡平均日增重、平均日采食量及显著增加料重比(P<0.05),并且地塞米松可显著降低肉仔鸡血浆内源皮质酮含量(P<0.05)。
试验二旨在研究地塞米松对肉仔鸡离体空肠外翻肠囊吸收转运Gly-Sar及空肠黏膜形态的影响。本试验以试验一中肉仔鸡为试验材料,28日龄时,每个重复取4只鸡,屠宰后取每只鸡相同部位空肠肠段,制备外翻肠囊及肠道组织切片。与对照组相比,地塞米松可显著降低空肠囊膜Gly-Sar的吸收量(P<0.05),显著降低空肠绒毛高度、吸收面积和绒毛高度/隐窝深度值(P<0.05),显著增加隐窝深度(P<0.05),但对空肠绒毛宽度无显著影响(P>0.05)。
试验三旨在研究地塞米松对肉仔鸡空肠上皮细胞小肽转运载体PepT-1mRNA表达量及BBMV转运Gly-Sar能力的影响。本试验以试验一中肉仔鸡为试验材料,28日龄时,每个重复取4只鸡,屠宰后取每只鸡相同部位空肠肠段,制备BBMV及用作实时定量检测。与对照组相比,地塞米松可显著降低肉仔鸡空肠上皮细胞小肽转运载体PepT-1mRNA相对表达量(P<0.05),显著降低BBMV Gly-Sar的转运能力(P<0.05)。
试验四旨在体外培养肉仔鸡原代肝脏细胞以提供试验模型。本试验以健康的21日龄雄性AA肉仔鸡为试验材料,采用原位灌注法分离培养肉仔鸡原代肝脏细胞。结果表明,应用原位灌注法分离培养的肉仔鸡肝脏细胞培养8天后,仍可维持正常的形态及功能,可满足后续试验需要。
试验五旨在研究酪啡肽(β-CM-3)对肉仔鸡体外培养的原代肝脏细胞脂肪代谢的影响。本试验以体外培养的肉仔鸡原代肝脏细胞为试验材料,将培养三天的肝脏原代细胞以1×105个/孔浓度接种于24孔板,培养一天后,向细胞培养板加入浓度分别为0,0.1,0.01,0.001,0.0001,0.00001 nmol/mlβ-CM-3,每个浓度作用时间为0,2,4,6,8h,每个时间及浓度对应三个重复。结果表明,与对照组相比β-CM-3可显著增加肉仔鸡肝脏细胞裂解液中脂肪酸合成酶、甘油三酯、丙二酰-CoA浓度(P<0.05)及显著降低肉毒羧碱转移酶浓度(P<0.05),β-CM-3处理时间及处理浓度间具有显著交互作用(P<0.05)。此外,纳洛酮可显著抑制β-CM-3上述作用(P<0.05)。
试验六旨在研究β-CM-3调控肉仔鸡体外培养原代肝脏细胞脂肪代谢的信号传导途径。本试验以体外培养的肉仔鸡原代肝脏细胞为试验材料,将培养三天的肝脏原代细胞以1×105个/孔浓度接种于24孔板,培养一天后,向细胞培养板中分别加入0.1nmol/mlβ-CM-3,0.1nmol/mlβ-CM-3+0.2nmol/ml PD98059,处理8h,每个处理3个重复。结果表明,与对照组相比,β-CM-3可显著增加肉仔鸡肝脏细胞裂解液中LXR浓度(P<0.05),以及磷酸化的Raf、ERK1/2、MEK浓度(P<0.05);与对照相比0.1nmol/mlβ-CM-3可显著增加肉仔鸡肝脏细胞裂解液中脂肪酸合成酶、丙二酰-CoA、甘油三酯浓度(P<0.05),并显著降低肉仔鸡肝脏细胞裂解液中肉毒羧碱转移酶含量(P<0.05)。抑制剂PD98059可显著阻断β-CM-3上述作用(P<0.05)。
试验七旨在研究β-CM-3对肉仔鸡脂肪代谢的影响。本试验选取体重相近的21日龄雄性AA肉仔鸡80只,随机分为4个处理,每个处理4个重复,每个重复5只鸡。其中3个处理组注射β-CM-3,注射剂量分别为0.1、0.5、1.0mg/kgBW,对照组注射等体积的生理盐水,连续注射7天。结果表明,对照组相比,β-CM-3可显著提高肉仔鸡平均日增重、平均日采食量及显著降低料重比(P<0.05),显著提高肝脏、胸肌脂肪含量及腹脂率(P<0.05);与对照组相比β-CM-3对胸肌中激素敏感脂肪酶(HSL)活性影响不显著(P>0.05),但可显著降低肝脏及腹脂中HSL活性(P<0.05)及显著升高胸肌、肝脏及腹脂中苹果酸脱氢酶(MDH)活性(P<0.05);与对照组相比,β-CM-3可显著提高血浆中胰岛素含量(P<0.05),可显著减低血浆中胰高血糖素的含量(P<0.05)以及显著提高肉仔鸡血浆中极低密度脂蛋白和甘油三酯的含量(P<0.05),但对甲状腺素及生长激素含量影响不显著(P>0.05)。
试验八旨在筛选与β-CM-3调控肉仔鸡脂肪代谢相关的基因。本试验应用Affymetrix鸡基因组芯片,筛选β-CM-3处理前后与肉仔鸡肝脏脂肪代谢相关的表达量差异在4倍以上的基因。结果表明,β-CM-3处理前后肉仔鸡肝脏表达量差异在4倍以上的基因共168个,其中与脂肪代谢相关基因共37个。
The aim of this study was to investigate the effects of glucocorticoids on the absorption of small peptides and the effects of small peptides on fat metabolism of broilers using Gly-Sar and casmorphin (β-CM-3) respectively.
Exp.1 The aim of this study was to investigate the effects of DEX on the performance and endogenous plasma corticosterone of broilers. Two hundreds 21-day-old male broilers of similar BW were chosen and randomly divided into four treatments, and were injected subcutaneously in the abdomen with DEX for 7d. Each treatment has five replicates ( 10 chicks per replicate), including three DEX treatments groups (0.1, 0.5, and 2.5 mg/kg BW) and one control group (saline injection).The results showed that, DEX significantly decreased average daily gain、average daily intake and increased feed conversation ratio of broilers compared with that of the control(P<0.05), and DEX led to a significant decrease in endogenous plasma corticosterone compared with that of the control(P<0.05).
Exp.2 The aim of this study was to investigate the effects of DEX on Gly-Sar absorption in jejunum everted gut sacs and jejunum mucosal morphology of broilers. In this study, the 28-day-old broilers from Exp.1 were used as the test materials after slaughtering, and the same segment of jejunum was removed from each chick (four chicks per treatment ) to prepare for the everted gut sac and intestinal tissue slice. DEX led to a significant decrease in the absorption of Gly-Sar transport into jejunum everted gut sacs compared with that of the contro(lP<0.05). DEX treatment led to a significant decrease in villus height, mucosal absorption area, and the ratio of villus height to crypt depth and led to an increase in the crypt depth compared with that of the control (P<0.05), however, the villus width of broilers was not significantly different between the control and treatment groups (P>0.05).
Exp.3 The aim of this study was to investigate the effects of DEX on mRNA transcription of cPepT-1 in the jejunum epithelium and Gly-Sar uptake in BBMV of broilers. In this study, the 28-day-old broilers from Exp.1 were used as the test materials after slaughtering, and the same segment of jejunum was removed from each chick (four chicks per treatment) and was prepared for the BBMV and real time PCR. DEX significantly decreased the cPepT-1 mRNA expression compared with that of the control (P<0.05), and DEX significantly decreased the rate of Gly-Sar transport into BBMV (P<0.05).
Exp.4 The aim of this study was to culture primary hepatocytes of broilers in vitro for the test model. The health 21-day-old male broilers were used as the test materials, and the hepatocytes of broilers were isolated and cultured by situ perfusion. The results showed that, the hepatocytes of broilers maintained the normal morphology and function after culturing for 8 days, so as to satisfy the need of the research.
Exp.5 The aim of this study was to investigate the effects ofβ-CM-3 on the fat metabolism of primary hepatocytes of broilers in vitro. The primary hepatocytes of broilers were used as the test materials. The hepatocytes (1×105 cells/plate) were inoculated in 24-well plates, the different concentrationβ-CM-3 were added into the plates(0,0.1,0.01,0.001,0.0001,0.00001 nmol/ml), and treating time were 0,2,4,6,8h respectively(3 replicates per concentration and time ) after culturing for 1 day. The results showed that,β-CM-3 significantly decreased the concentration of fatty acid synthase, triglyceride and malonyl-COA in the hepatocytes lysis solution of broilers compared with the control(P<0.05),and significantly increased the concentration of carnitine palmitoyl transferase(P<0.05). There was significant interaction between treatment concentration and time ofβ-CM-3(P<0.05).In addition, the naloxone significantly inhibited the above-mentioned functions ofβ-CM-3.
Exp.6 The aim of this study was to investigate the signal transduction pathway by whichβ-CM-3 regulated the fat metabolism of primary hepatocytes of broilers. The hepatocytes (1×105cells/plate) were inoculated in 24-well plates, the 0.1nmol/mlβ-CM-3 and 0.1nmol/mlβ-CM-3+0.2nmol/ml PD98059 were added in the plates, and treating time was 8h (3 replicates per treatment) after culturing for 1 day. The results showed that,β-CM-3 significantly decreased the concentration of LXR、Raf-P、ERK1/2-P and MEK-P compared with the control (P<0.05) .Theβ-CM-3 significantly decreased the concentration of fatty acid synthase, triglyceride and malonyl-COA in the hepatocytes lysis solution of broilers compared with the contro(lP<0.05), and significantly increased the concentration of carnitine palmitoyl transferase(P<0.05).In addition, the PD98059 significantly inhibited the above-mentioned functions ofβ-CM-3.
Exp.7 The aim of this study was to investigate the effects ofβ-CM-3 on the fat metabolism of broilers. 80 21-day-old male broilers of similar BW were chosen and randomly divided into four treatments, and were injected subcutaneously in the abdomen withβ-CM-3 for 7d. Each treatment has five replicates (10 chicks per replicate), including threeβ-CM-3 treatment groups (0.1, 0.5, and 1.0 mg/kg BW) and one control group (saline injection).The results showed that,β-CM-3 significantly increased average daily gain、average daily intake and decreased feed conversation ratio of broilers compared with that of the control(P<0.05); Theβ-CM-3 significantly increased the fat content of liver and breast muscle, and the percentage of abdominal fat compared with that of the control(P<0.05).Theβ-CM-3 did not significantly effect the activity of HSL in the breast muscl(eP>0.05),however it significantly decreased the activity of HSL in the liver and abdominal fat compared with that of the control(P<0.05); Theβ-CM-3 significantly increased the activity of MDH in the breast muscle、liver and abdominal fat compared with that of the control(P<0.05); Theβ-CM-3 significantly increased the concentration of insulin in the plasma, and significantly decreased the concentration of glucagon in the plasma compared with that of the control(P<0.05), however it did not significantly effect the concentration of thyroxin and growth hormon(eP>0.05); Theβ-CM-3 significantly increased the concentration of very low density lipoprotein and triglyceride in the plasma compared with that of the control (P<0.05).
Exp.8 The aim of this study was to select the genes related to fat metabolism of the broilers, which was controlled byβ-CM-3.The chicken gene-chips (Affymetrix) were used to select the differentially expressed by more than 4 times genes of broiler livers between preprocessing and postprocessing ofβ-CM-3. The results showed that, a total of 168 genes were differentially expressed in the liver of broiler including 37 genes related to fat metabolism.
试验一旨在研究糖皮质激素类物质地塞米松对肉仔鸡生产性能及血浆内源皮质酮含量的影响。试验选取体重相近的21日龄雄性AA肉仔鸡200只,随机分为4个处理,每个处理5个重复,每个重复10只鸡。其中3个处理组腹部皮下注射地塞米松,注射剂量分别为0.1、0.5、2.5mg/kgBW,对照组注射等体积生理盐水,连续注射7天。结果表明,与对照组相比地塞米松显著降低肉仔鸡平均日增重、平均日采食量及显著增加料重比(P<0.05),并且地塞米松可显著降低肉仔鸡血浆内源皮质酮含量(P<0.05)。
试验二旨在研究地塞米松对肉仔鸡离体空肠外翻肠囊吸收转运Gly-Sar及空肠黏膜形态的影响。本试验以试验一中肉仔鸡为试验材料,28日龄时,每个重复取4只鸡,屠宰后取每只鸡相同部位空肠肠段,制备外翻肠囊及肠道组织切片。与对照组相比,地塞米松可显著降低空肠囊膜Gly-Sar的吸收量(P<0.05),显著降低空肠绒毛高度、吸收面积和绒毛高度/隐窝深度值(P<0.05),显著增加隐窝深度(P<0.05),但对空肠绒毛宽度无显著影响(P>0.05)。
试验三旨在研究地塞米松对肉仔鸡空肠上皮细胞小肽转运载体PepT-1mRNA表达量及BBMV转运Gly-Sar能力的影响。本试验以试验一中肉仔鸡为试验材料,28日龄时,每个重复取4只鸡,屠宰后取每只鸡相同部位空肠肠段,制备BBMV及用作实时定量检测。与对照组相比,地塞米松可显著降低肉仔鸡空肠上皮细胞小肽转运载体PepT-1mRNA相对表达量(P<0.05),显著降低BBMV Gly-Sar的转运能力(P<0.05)。
试验四旨在体外培养肉仔鸡原代肝脏细胞以提供试验模型。本试验以健康的21日龄雄性AA肉仔鸡为试验材料,采用原位灌注法分离培养肉仔鸡原代肝脏细胞。结果表明,应用原位灌注法分离培养的肉仔鸡肝脏细胞培养8天后,仍可维持正常的形态及功能,可满足后续试验需要。
试验五旨在研究酪啡肽(β-CM-3)对肉仔鸡体外培养的原代肝脏细胞脂肪代谢的影响。本试验以体外培养的肉仔鸡原代肝脏细胞为试验材料,将培养三天的肝脏原代细胞以1×105个/孔浓度接种于24孔板,培养一天后,向细胞培养板加入浓度分别为0,0.1,0.01,0.001,0.0001,0.00001 nmol/mlβ-CM-3,每个浓度作用时间为0,2,4,6,8h,每个时间及浓度对应三个重复。结果表明,与对照组相比β-CM-3可显著增加肉仔鸡肝脏细胞裂解液中脂肪酸合成酶、甘油三酯、丙二酰-CoA浓度(P<0.05)及显著降低肉毒羧碱转移酶浓度(P<0.05),β-CM-3处理时间及处理浓度间具有显著交互作用(P<0.05)。此外,纳洛酮可显著抑制β-CM-3上述作用(P<0.05)。
试验六旨在研究β-CM-3调控肉仔鸡体外培养原代肝脏细胞脂肪代谢的信号传导途径。本试验以体外培养的肉仔鸡原代肝脏细胞为试验材料,将培养三天的肝脏原代细胞以1×105个/孔浓度接种于24孔板,培养一天后,向细胞培养板中分别加入0.1nmol/mlβ-CM-3,0.1nmol/mlβ-CM-3+0.2nmol/ml PD98059,处理8h,每个处理3个重复。结果表明,与对照组相比,β-CM-3可显著增加肉仔鸡肝脏细胞裂解液中LXR浓度(P<0.05),以及磷酸化的Raf、ERK1/2、MEK浓度(P<0.05);与对照相比0.1nmol/mlβ-CM-3可显著增加肉仔鸡肝脏细胞裂解液中脂肪酸合成酶、丙二酰-CoA、甘油三酯浓度(P<0.05),并显著降低肉仔鸡肝脏细胞裂解液中肉毒羧碱转移酶含量(P<0.05)。抑制剂PD98059可显著阻断β-CM-3上述作用(P<0.05)。
试验七旨在研究β-CM-3对肉仔鸡脂肪代谢的影响。本试验选取体重相近的21日龄雄性AA肉仔鸡80只,随机分为4个处理,每个处理4个重复,每个重复5只鸡。其中3个处理组注射β-CM-3,注射剂量分别为0.1、0.5、1.0mg/kgBW,对照组注射等体积的生理盐水,连续注射7天。结果表明,对照组相比,β-CM-3可显著提高肉仔鸡平均日增重、平均日采食量及显著降低料重比(P<0.05),显著提高肝脏、胸肌脂肪含量及腹脂率(P<0.05);与对照组相比β-CM-3对胸肌中激素敏感脂肪酶(HSL)活性影响不显著(P>0.05),但可显著降低肝脏及腹脂中HSL活性(P<0.05)及显著升高胸肌、肝脏及腹脂中苹果酸脱氢酶(MDH)活性(P<0.05);与对照组相比,β-CM-3可显著提高血浆中胰岛素含量(P<0.05),可显著减低血浆中胰高血糖素的含量(P<0.05)以及显著提高肉仔鸡血浆中极低密度脂蛋白和甘油三酯的含量(P<0.05),但对甲状腺素及生长激素含量影响不显著(P>0.05)。
试验八旨在筛选与β-CM-3调控肉仔鸡脂肪代谢相关的基因。本试验应用Affymetrix鸡基因组芯片,筛选β-CM-3处理前后与肉仔鸡肝脏脂肪代谢相关的表达量差异在4倍以上的基因。结果表明,β-CM-3处理前后肉仔鸡肝脏表达量差异在4倍以上的基因共168个,其中与脂肪代谢相关基因共37个。
The aim of this study was to investigate the effects of glucocorticoids on the absorption of small peptides and the effects of small peptides on fat metabolism of broilers using Gly-Sar and casmorphin (β-CM-3) respectively.
Exp.1 The aim of this study was to investigate the effects of DEX on the performance and endogenous plasma corticosterone of broilers. Two hundreds 21-day-old male broilers of similar BW were chosen and randomly divided into four treatments, and were injected subcutaneously in the abdomen with DEX for 7d. Each treatment has five replicates ( 10 chicks per replicate), including three DEX treatments groups (0.1, 0.5, and 2.5 mg/kg BW) and one control group (saline injection).The results showed that, DEX significantly decreased average daily gain、average daily intake and increased feed conversation ratio of broilers compared with that of the control(P<0.05), and DEX led to a significant decrease in endogenous plasma corticosterone compared with that of the control(P<0.05).
Exp.2 The aim of this study was to investigate the effects of DEX on Gly-Sar absorption in jejunum everted gut sacs and jejunum mucosal morphology of broilers. In this study, the 28-day-old broilers from Exp.1 were used as the test materials after slaughtering, and the same segment of jejunum was removed from each chick (four chicks per treatment ) to prepare for the everted gut sac and intestinal tissue slice. DEX led to a significant decrease in the absorption of Gly-Sar transport into jejunum everted gut sacs compared with that of the contro(lP<0.05). DEX treatment led to a significant decrease in villus height, mucosal absorption area, and the ratio of villus height to crypt depth and led to an increase in the crypt depth compared with that of the control (P<0.05), however, the villus width of broilers was not significantly different between the control and treatment groups (P>0.05).
Exp.3 The aim of this study was to investigate the effects of DEX on mRNA transcription of cPepT-1 in the jejunum epithelium and Gly-Sar uptake in BBMV of broilers. In this study, the 28-day-old broilers from Exp.1 were used as the test materials after slaughtering, and the same segment of jejunum was removed from each chick (four chicks per treatment) and was prepared for the BBMV and real time PCR. DEX significantly decreased the cPepT-1 mRNA expression compared with that of the control (P<0.05), and DEX significantly decreased the rate of Gly-Sar transport into BBMV (P<0.05).
Exp.4 The aim of this study was to culture primary hepatocytes of broilers in vitro for the test model. The health 21-day-old male broilers were used as the test materials, and the hepatocytes of broilers were isolated and cultured by situ perfusion. The results showed that, the hepatocytes of broilers maintained the normal morphology and function after culturing for 8 days, so as to satisfy the need of the research.
Exp.5 The aim of this study was to investigate the effects ofβ-CM-3 on the fat metabolism of primary hepatocytes of broilers in vitro. The primary hepatocytes of broilers were used as the test materials. The hepatocytes (1×105 cells/plate) were inoculated in 24-well plates, the different concentrationβ-CM-3 were added into the plates(0,0.1,0.01,0.001,0.0001,0.00001 nmol/ml), and treating time were 0,2,4,6,8h respectively(3 replicates per concentration and time ) after culturing for 1 day. The results showed that,β-CM-3 significantly decreased the concentration of fatty acid synthase, triglyceride and malonyl-COA in the hepatocytes lysis solution of broilers compared with the control(P<0.05),and significantly increased the concentration of carnitine palmitoyl transferase(P<0.05). There was significant interaction between treatment concentration and time ofβ-CM-3(P<0.05).In addition, the naloxone significantly inhibited the above-mentioned functions ofβ-CM-3.
Exp.6 The aim of this study was to investigate the signal transduction pathway by whichβ-CM-3 regulated the fat metabolism of primary hepatocytes of broilers. The hepatocytes (1×105cells/plate) were inoculated in 24-well plates, the 0.1nmol/mlβ-CM-3 and 0.1nmol/mlβ-CM-3+0.2nmol/ml PD98059 were added in the plates, and treating time was 8h (3 replicates per treatment) after culturing for 1 day. The results showed that,β-CM-3 significantly decreased the concentration of LXR、Raf-P、ERK1/2-P and MEK-P compared with the control (P<0.05) .Theβ-CM-3 significantly decreased the concentration of fatty acid synthase, triglyceride and malonyl-COA in the hepatocytes lysis solution of broilers compared with the contro(lP<0.05), and significantly increased the concentration of carnitine palmitoyl transferase(P<0.05).In addition, the PD98059 significantly inhibited the above-mentioned functions ofβ-CM-3.
Exp.7 The aim of this study was to investigate the effects ofβ-CM-3 on the fat metabolism of broilers. 80 21-day-old male broilers of similar BW were chosen and randomly divided into four treatments, and were injected subcutaneously in the abdomen withβ-CM-3 for 7d. Each treatment has five replicates (10 chicks per replicate), including threeβ-CM-3 treatment groups (0.1, 0.5, and 1.0 mg/kg BW) and one control group (saline injection).The results showed that,β-CM-3 significantly increased average daily gain、average daily intake and decreased feed conversation ratio of broilers compared with that of the control(P<0.05); Theβ-CM-3 significantly increased the fat content of liver and breast muscle, and the percentage of abdominal fat compared with that of the control(P<0.05).Theβ-CM-3 did not significantly effect the activity of HSL in the breast muscl(eP>0.05),however it significantly decreased the activity of HSL in the liver and abdominal fat compared with that of the control(P<0.05); Theβ-CM-3 significantly increased the activity of MDH in the breast muscle、liver and abdominal fat compared with that of the control(P<0.05); Theβ-CM-3 significantly increased the concentration of insulin in the plasma, and significantly decreased the concentration of glucagon in the plasma compared with that of the control(P<0.05), however it did not significantly effect the concentration of thyroxin and growth hormon(eP>0.05); Theβ-CM-3 significantly increased the concentration of very low density lipoprotein and triglyceride in the plasma compared with that of the control (P<0.05).
Exp.8 The aim of this study was to select the genes related to fat metabolism of the broilers, which was controlled byβ-CM-3.The chicken gene-chips (Affymetrix) were used to select the differentially expressed by more than 4 times genes of broiler livers between preprocessing and postprocessing ofβ-CM-3. The results showed that, a total of 168 genes were differentially expressed in the liver of broiler including 37 genes related to fat metabolism.
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