维生素E对蛋鸡维生素A关键转运蛋白和分解酶的影响
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摘要
本论文包括体内和体外两个试验。探讨了维生素E对维生素A的影响及其作用机制。
试验一维生素E对蛋鸡体内维生素A关键转运蛋白和分解酶的影响
选取体重和产蛋率相近的24周龄京红产蛋鸡450只,随机分为5个处理组,每处理设6个重复,每个重复15只鸡,分别采食基础日粮和4种试验日粮。基础日粮不添加VE,4种试验日粮分别添加20、80、320和1280 IU/kg VE。预饲期4周,试验期8周。结果表明:在试验第4和第8周,随着日粮VE添加量的增加,肝脏中VA的含量显著增加(P<0.05)且呈现剂量效应。日粮中高剂量的VE(320 IU/kg和1280 IU/kg)可显著降低肝脏RBP的含量(P<0.05)。蛋鸡肝脏微粒体蛋白和CYP450含量不受日粮VE添加量的影响(P>0.05)。
试验二蛋鸡肝细胞分离及制备方法的优化
本试验通过两种肝细胞分离及制备方法的对比,旨在对蛋鸡肝脏细胞分离和制备方法进行优化。以结扎肠系膜后静脉远心端,不结扎输卵管静脉和胰十二指肠静脉的成年鸡肝细胞培养灌流法为对照组;以本试验建立的同时结扎肠系膜后静脉远心端、输卵管静脉和胰十二指肠静脉的灌流方法为处理组,进行蛋鸡肝细胞培养,分别记录肝脏灌流的成功率、肝细胞数量和细胞活率。同时,分别于试验第4 h、24 h、48 h、144 h和168 h观察细胞形态、测定肝细胞培养基上清液中乳酸脱氢酶(LDH)活力。结果表明,优化后的方法可使蛋鸡肝脏灌流的成功率提高至100%,肝细胞数量显著增加至(1.09±0.21)×109个(P <0.05),细胞活率也显著提升至(95.3±1.3)% (P <0.05)。两种方法培养的肝细胞形态和培养基上清液LDH活力无显著差异(P >0.05)。结果表明,本试验建立的肝细胞分离和制备方法比原有方法,更适用于产蛋家禽肝细胞的分离和制备,通过此法可获得量多、质优的肝细胞,满足相关试验研究需要。
试验三α-生育酚对蛋鸡肝细胞视黄醇结合蛋白和细胞色素P450酶系主要亚型的影响
本试验以蛋鸡肝细胞为试验对象,按照细胞培养液中添加的α-生育酚浓度(0、10、50和100μM)不同分为4个处理。将用于肝细胞原代培养的1只产蛋鸡设为1个重复,每个处理设4个重复,每个重复设6个培养孔。α-生育酚处理20 min后收集细胞培养液和细胞悬液,测定其LDH、总蛋白和RBP含量;并用半定量PCR法测定肝细胞RBP、CYP26A1的mRNA水平。结果表明:α-生育酚100μM可显著降低肝细胞RBP的含量及其mRNA的表达量(P<0.05)。α-生育酚(0,10,50,100μM)对CYP450酶系主要亚型CYP26A1的m RNA表达量无显著影响(P>0.05)。
This thesis includes vivo and vitro two parts of experiments. We investigated the effect of vitamin E on key transporter and catabolic enzyme of vitamin A in laying hens, and we used hepatocytes from laying hens to test the effect ofα-tocopherol on the retinol binding protein and the major subtypes of cytochrome P450 enzymes.
Experiment 1 The Effect of Vitamin E on Key Transporter and Catabolic Enzyme of Vitamin A in Laying Hens
Four hundred and fifty JingHong Brown hens (24 weeks old) were randomly divided to five treatments with six replicates .The control group with fifteen hens in each replicate received corn-soybean meal diet without vitamin E, and the other groups received the same basal diet supplemented vitamin E with 20, 80, 320, 1280 IU/kg respectively. The feeding trail lasted eight weeks.Production performance was recorded daily throughout the experimental period. Feed consumption and feed conversion were measured in each week. Eggs in each replicate were collected and the shell hardness, Haugh units, yolk color, shell thickness and albumen height were measured each 2 weeks. Vitamin E and A in feed were determined at the beginning of the experiment. In 4th and 8th week all egg yolks in each replicate were collected and slaughtered 1 bird in each replicate to get liver and plasma to determined vitamin E, vitamin A. Analysis of RBP and CYP450 in liver was carried out.The result showed that in the 4th week and 8th week, adding different levels of vitamin E in dietary, the content of retinol(and retinol palmitate)was significantly increased (P<0.05) in liver. In the 8th week, higher vitamin E (320, 1280 IU/kg) supplementations decreased vitamin A concentrations of egg yolk significantly (P<0.05).The plasma concentration of retinol was independent of the vitamin E supply. RBP in 320 and 1280 IU/kg dietary vitamin E groups was significantly (P<0.05) depressed in contrast to control group. Supplementation with vitamin E in either group had no detectable effect on plasma RBP concentration over the experimental period. No differences were observed in the content of CYP450.
Experiment 2 Optimization of the Methods for Isolation and Preparation of Hepatocytes in Laying Hens
This study was to compared two methods of isolation and preparation of hepatocytes, in order to optimize the methods of hepatocytes culture in laying hens.The control group was used the method that ligated posterior mesenteric vein in distal end and free tubal vein as well as pancreaticoduodenal vein. The treatment group was used the method that ligated posterior mesenteric vein in distal end, tubal vein and pancreaticoduodenal vein. The success rate of liver perfusion, total hepatocytes yield and cell viability was recorded during the experiments. Meanwhile, the cell morphology was observed and hepatic lactate dehydrogenase (LDH) activity was determined in 4, 24, 48, 144 and 168 h. After optimizing the method, the success rate of liver perfusion was increased to 100%, and the total hepatocytes yield and cell viability were significantly increased to (1.09±0.21)×109 per liver (P <0.05) and (95.3±1.3)% (P <0.05) respectively. However, there was no significant difference between two methods in the cell morphology and LDH activity (P >0.05).These results indicated that in compared with the old method, the method for isolation and preparation of hepatocytes that established in this experiment was more suitable for hepatocytes culture in egg laying poultry, which can obtain high quality of hepatocytes and meet the requirement of the further study.
Experiment 3 The Effect ofα-tocopherol on Retinol Binding Protein and CYP26A1 in Hepatocytes from Laying Hens
The laying hens were feed vitamin E free dietary as Experiment 1 described. The methods and materials used in hepatocytes isolation and culture procedures were according to those described in Experimat 2. The culture conditions were identical with normal hepatocytes, and the initial medium was replaced withα-tocopherol (0, 10, 50, 100μM)supplemented William’s medium E(serum-free) after 5 hr in culture.Cells were harvested by 20 minutes after addition ofα-tocopherol to the medium. The lactate dehydrogenase activity, total protein and RBP were determined after 20 min. Analysis of RBP and CYP26A1 expression at the mRNA level were used the technology of reverse transcriptase-polymerase chain reaction (RT-PCR).α-tocopherol on LDH activity, total protein had no significant effect (P>0.05).α-tocopherol 100μM group significantly reduced the content of RBP and its mRNA expression (P<0.05). There was no significant effect on CYP26A1 mRNA expression.
试验一维生素E对蛋鸡体内维生素A关键转运蛋白和分解酶的影响
选取体重和产蛋率相近的24周龄京红产蛋鸡450只,随机分为5个处理组,每处理设6个重复,每个重复15只鸡,分别采食基础日粮和4种试验日粮。基础日粮不添加VE,4种试验日粮分别添加20、80、320和1280 IU/kg VE。预饲期4周,试验期8周。结果表明:在试验第4和第8周,随着日粮VE添加量的增加,肝脏中VA的含量显著增加(P<0.05)且呈现剂量效应。日粮中高剂量的VE(320 IU/kg和1280 IU/kg)可显著降低肝脏RBP的含量(P<0.05)。蛋鸡肝脏微粒体蛋白和CYP450含量不受日粮VE添加量的影响(P>0.05)。
试验二蛋鸡肝细胞分离及制备方法的优化
本试验通过两种肝细胞分离及制备方法的对比,旨在对蛋鸡肝脏细胞分离和制备方法进行优化。以结扎肠系膜后静脉远心端,不结扎输卵管静脉和胰十二指肠静脉的成年鸡肝细胞培养灌流法为对照组;以本试验建立的同时结扎肠系膜后静脉远心端、输卵管静脉和胰十二指肠静脉的灌流方法为处理组,进行蛋鸡肝细胞培养,分别记录肝脏灌流的成功率、肝细胞数量和细胞活率。同时,分别于试验第4 h、24 h、48 h、144 h和168 h观察细胞形态、测定肝细胞培养基上清液中乳酸脱氢酶(LDH)活力。结果表明,优化后的方法可使蛋鸡肝脏灌流的成功率提高至100%,肝细胞数量显著增加至(1.09±0.21)×109个(P <0.05),细胞活率也显著提升至(95.3±1.3)% (P <0.05)。两种方法培养的肝细胞形态和培养基上清液LDH活力无显著差异(P >0.05)。结果表明,本试验建立的肝细胞分离和制备方法比原有方法,更适用于产蛋家禽肝细胞的分离和制备,通过此法可获得量多、质优的肝细胞,满足相关试验研究需要。
试验三α-生育酚对蛋鸡肝细胞视黄醇结合蛋白和细胞色素P450酶系主要亚型的影响
本试验以蛋鸡肝细胞为试验对象,按照细胞培养液中添加的α-生育酚浓度(0、10、50和100μM)不同分为4个处理。将用于肝细胞原代培养的1只产蛋鸡设为1个重复,每个处理设4个重复,每个重复设6个培养孔。α-生育酚处理20 min后收集细胞培养液和细胞悬液,测定其LDH、总蛋白和RBP含量;并用半定量PCR法测定肝细胞RBP、CYP26A1的mRNA水平。结果表明:α-生育酚100μM可显著降低肝细胞RBP的含量及其mRNA的表达量(P<0.05)。α-生育酚(0,10,50,100μM)对CYP450酶系主要亚型CYP26A1的m RNA表达量无显著影响(P>0.05)。
This thesis includes vivo and vitro two parts of experiments. We investigated the effect of vitamin E on key transporter and catabolic enzyme of vitamin A in laying hens, and we used hepatocytes from laying hens to test the effect ofα-tocopherol on the retinol binding protein and the major subtypes of cytochrome P450 enzymes.
Experiment 1 The Effect of Vitamin E on Key Transporter and Catabolic Enzyme of Vitamin A in Laying Hens
Four hundred and fifty JingHong Brown hens (24 weeks old) were randomly divided to five treatments with six replicates .The control group with fifteen hens in each replicate received corn-soybean meal diet without vitamin E, and the other groups received the same basal diet supplemented vitamin E with 20, 80, 320, 1280 IU/kg respectively. The feeding trail lasted eight weeks.Production performance was recorded daily throughout the experimental period. Feed consumption and feed conversion were measured in each week. Eggs in each replicate were collected and the shell hardness, Haugh units, yolk color, shell thickness and albumen height were measured each 2 weeks. Vitamin E and A in feed were determined at the beginning of the experiment. In 4th and 8th week all egg yolks in each replicate were collected and slaughtered 1 bird in each replicate to get liver and plasma to determined vitamin E, vitamin A. Analysis of RBP and CYP450 in liver was carried out.The result showed that in the 4th week and 8th week, adding different levels of vitamin E in dietary, the content of retinol(and retinol palmitate)was significantly increased (P<0.05) in liver. In the 8th week, higher vitamin E (320, 1280 IU/kg) supplementations decreased vitamin A concentrations of egg yolk significantly (P<0.05).The plasma concentration of retinol was independent of the vitamin E supply. RBP in 320 and 1280 IU/kg dietary vitamin E groups was significantly (P<0.05) depressed in contrast to control group. Supplementation with vitamin E in either group had no detectable effect on plasma RBP concentration over the experimental period. No differences were observed in the content of CYP450.
Experiment 2 Optimization of the Methods for Isolation and Preparation of Hepatocytes in Laying Hens
This study was to compared two methods of isolation and preparation of hepatocytes, in order to optimize the methods of hepatocytes culture in laying hens.The control group was used the method that ligated posterior mesenteric vein in distal end and free tubal vein as well as pancreaticoduodenal vein. The treatment group was used the method that ligated posterior mesenteric vein in distal end, tubal vein and pancreaticoduodenal vein. The success rate of liver perfusion, total hepatocytes yield and cell viability was recorded during the experiments. Meanwhile, the cell morphology was observed and hepatic lactate dehydrogenase (LDH) activity was determined in 4, 24, 48, 144 and 168 h. After optimizing the method, the success rate of liver perfusion was increased to 100%, and the total hepatocytes yield and cell viability were significantly increased to (1.09±0.21)×109 per liver (P <0.05) and (95.3±1.3)% (P <0.05) respectively. However, there was no significant difference between two methods in the cell morphology and LDH activity (P >0.05).These results indicated that in compared with the old method, the method for isolation and preparation of hepatocytes that established in this experiment was more suitable for hepatocytes culture in egg laying poultry, which can obtain high quality of hepatocytes and meet the requirement of the further study.
Experiment 3 The Effect ofα-tocopherol on Retinol Binding Protein and CYP26A1 in Hepatocytes from Laying Hens
The laying hens were feed vitamin E free dietary as Experiment 1 described. The methods and materials used in hepatocytes isolation and culture procedures were according to those described in Experimat 2. The culture conditions were identical with normal hepatocytes, and the initial medium was replaced withα-tocopherol (0, 10, 50, 100μM)supplemented William’s medium E(serum-free) after 5 hr in culture.Cells were harvested by 20 minutes after addition ofα-tocopherol to the medium. The lactate dehydrogenase activity, total protein and RBP were determined after 20 min. Analysis of RBP and CYP26A1 expression at the mRNA level were used the technology of reverse transcriptase-polymerase chain reaction (RT-PCR).α-tocopherol on LDH activity, total protein had no significant effect (P>0.05).α-tocopherol 100μM group significantly reduced the content of RBP and its mRNA expression (P<0.05). There was no significant effect on CYP26A1 mRNA expression.
引文
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