维生素A过量对肉鸡钙、磷沉积及碱性磷酸酶和骨钙素表达的影响
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摘要
本论文共分4个试验研究了维生素A过量对肉鸡钙、磷沉积及碱性磷酸和骨钙素基因表达的影响。试验1~试验3利用体内法,采用单因子完全随机试验设计,将1日龄健康爱拔益加(AA)肉鸡(公母各半)450只,随机分为5个日粮处理组,每个处理组6个重复,每个重复组15只鸡,试验期42天。以玉米和豆粕为主要原料配制基础日粮(基础日粮中不添加维生素A),5种试验日粮分别是在基础日粮中添加3000、6000、15000、30000和60000 IU/kg的维生素A配制而成。试验1通过研究过量维生素A对肉鸡血清钙、磷及胫骨钙、磷含量、胫骨BMD及胫骨灰分的影响,进一步验证过量维生素A对肉鸡钙、磷沉积的影响。试验2主要通过研究维生素A过量对肉鸡钙、磷沉积相关激素及骨形成标志物的影响,探讨了维生素A过量对肉鸡钙、磷沉积的影响机制。试验3主要通过研究维生素A过量对肉鸡胫骨、肝脏及小肠组织中ALP mRNA和BGP mRNA相对表达量的影响,探讨了过量维生素A对肉鸡钙、磷沉积影响的分子机理。试验4利用体外细胞培养法,研究了不同浓度维生素A(0,0.03,0.10,0.30,1.00,2.00,6.00,12.00,24.00μg/ml)对肉鸡颅骨成骨细胞分泌功能及ALP mRNA和BGP mRNA相对表达量的影响,从成骨细胞活性的角度在细胞水平和分子水平上进一步探讨了维生素A过量对肉鸡钙、磷沉积的影响机制。
在本研究的试验条件下初步得出以下结果:
①肉鸡胫骨钙、磷沉积和胫骨强度与日粮维生素A水平呈显著或趋于显著的剂量依赖关系(P<0.05;P<0.10)。其中,胫骨钙磷沉积与胫骨强度以3000~6000 IU/kg维生素A组较高;30000~60000 IU/kg维生素A组较低;日粮维生素A水平为15000 IU/kg时,胫骨钙、磷沉积与胫骨强度也呈一定降低趋势。
②日粮维生素A水平在3000~60000 IU/kg范围内,日粮维生素A水平以剂量依赖模式影响肉鸡的血清PTH、CT、1,25(OH)_2D_3、BGP与PINP浓度及血清ALP和BAP活性(P<0.05;P<0.10)。其中,30000~60000 IU/kg的高剂量维生素A引起肉鸡血清中PTH浓度升高,CT与1,25(OH)2D3浓度及BGP与PINP浓度降低,ALP与BAP活性降低。
③随日粮维生素A添加剂量的升高,肉鸡胫骨、肝脏及小肠组织中ALP mRNA和BGP mRNA相对表达量均呈不同程度降低,二者呈显著(P<0.05)或趋于显著(P<0.10)的一次线性或二次曲线剂量依赖关系;其表达量均以3000~6000 IU/kg维生素A日粮组较高,30000~60000 IU/kg维生素A日粮组较低,15000IU/kg维生素A日粮组居中。提示日粮维生素A过量可下调肉鸡胫骨和肝脏及小肠组织中ALP mRNA和BGP mRNA的相对表达量。
④随培养液中维生素A水平的增加,肉鸡成骨细胞培养液中ALP活性、BGP、PINP、OPN和OPG浓度呈显著的一次线性降低(P<0.05)或二次曲线降低趋势(P<0.10)。提示维生素A水平以剂量依赖模式影响肉鸡成骨细胞的功能和活性,适宜的维生素A水平可促进成骨细胞的活性,诱导其分化成熟,但高剂量的维生素A抑制成骨细胞的活性和分化成熟。
⑤随培养液中维生素A水平的增加,成骨细胞ALP mRNA相对表达量呈显著的一次线性或一元二次降低(P<0.05),BGP mRNA相对表达量呈一元二次降低趋势(P<0.10)。提示维生素A以剂量依赖模式影响成骨细胞ALP mRNA与BGP mRNA相对表达量,适宜剂量维生素A可上调其表达,高剂量维生素A则下调其表达,低剂量维生素A对BGP mRNA相对表达量也有一定抑制效果。
⑥综合试验1~4的结果,维生素A过量影响肉鸡钙磷沉积的分子机理可能是:过量维生素A抑制了组织和成骨细胞ALP mRNA和BGP mRNA的相对表达量,降低了ALP和BGP的合成,进而对骨骼代谢及钙、磷沉积造成负面影响。
The present study was conducted to investigate the effects of vitamin A excess on calcium and phosphorus deposition and alkaline phosphatase and bone gla-protein mRNA expression in broiler chickens. The first three experiments were conducted by using in vivo experimental techniques. A total of 450 one-day-old Arbor Acre half male and half female broiler chickens with similar body weight were randomly allotted to five dietary treatments with six replicate cages per dietary treatment and fifteen birds per cage. The basal diet that didn’t contain vitamin A was mainly composed of corn and soybean meal.The broiler chickens in the five treatments were fed the basal diets supplemented with 3000, 6000, 15000, 30000 or 60000 IU/kg vitamin A in a single factorial arrangement. The trial lasted for 42 days. The objective of the experiment 1 was to elucidate the effects of vitamin A excess on contents of Ca, P in serum and concentrations of ash, Ca and P and BMD in tibia of broilers, and further to demonstrate the negative effects of excessive vitamin A on calcium and phosphorus deposition in broiler chickens. The experiment 2 was conducted to investigate the effects of vitamin A excess on hormones related to calcium and phosphorus deposition and biochemical markers of bone formation. The experiment 3 was conducted to study the effects of vitamin A excess on relative expression of ALP mRNA and BGP mRNA in small intestine, tibia and liver, and to elucidate the molecular mechanisms for vitamin A excess to affect calcium and phosphorus deposition in broiler chickens. The experiment 4 was conducted to demonstrate the effects of different levels of vitamin A(0, 0.03, 0.10, 0.30, 1.00, 2.00, 6.00, 12.00, 24.00μg/ml) on osteoblasts in vitro by evaluating cells activity and relative expression of ALP mRNA and BGP mRNA of osteoblastic cells and further to investigate the molecular mechanisms by which vitamin A modulated calcium and phosphorus deposition.
Under the present experimental conditions, the results showed as follows:
①Dietary vitamin A influenced the strength and disposition of calcium and phosphours in the tibia of broilers in a dose-dependent manner(P<0.05;P<0.10). The supplementation of 3000~6000 IU/kg vitamin A in the diet improved the strength and disposition of calcium in the tibia. But the strength and disposition of calcium in the tibia tended to be suppressed when the addition of vitamin A in diet was increased to 30000~60000 IU/kg. And the tibia strength tended to be lower while 15000 IU/kg vitamin A was added in the diet.
②Dietary vitamin A influenced contents of PTH, CT, 1,25(OH)2D3, BGP and PINP as well as ALP and BAP activity in serum in a dose-dependent manner (P<0.05; P<0.10) with the supplementation of vitamin A in the diet from 3000 to 60000 IU/kg. The addition of 30000～60000 IU/kg vitamin A to the diet increased serum PTH level, and depressed the contents of CT, 1,25(OH)2D3, BGP and PINP as well as ALP and BAP activity in serum of broilers.
③The dietary vitamin A influenced the relative expressions of the ALP mRNA and BGP mRNA in the tibia, liver and small intestine in a linear or quadratical dose-dependent manner (P<0.05, P<0.10). The addition of 3 000~6 000 IU/kg vitamin A to the diet showed the better effect in relative expressions of ALP mRNA and BGP mRNA in tibia, liver and the small intestine of broilers. However, the addition of 3 0000~6 0000 IU/kg vitamin A to the diet could down-regulate the relative expressions of ALP mRNA and BGP mRNA, and the addition of 15 000 IU/kg vitamin A tended to decrease in relative expressions of ALP mRNA and BGP mRNA.
④With the increase of vitamin A in the culture, ALP activity and concentrations of BGP, PINP, OPN and OPG in the culture of the cranium osteoblasts of broilers decreased linearly or quadratically(P<0.05, P<0.10). This suggested that the vitamin A could promote the secretion functions and activity of cranium osteoblasts in broiler chickens in a dose-dependent manner. Adequate doses of vitamin A could promote the activity of osteoblasts, and induce differentiation and maturation. But the large doses of vitamin A was less effective.
⑤With the increase of vitamin A in the culture of the osteoblasts in broilers, the relative expression of the ALP mRNA of osteoblasts decreased linearly or quadratically(P<0.05), and the relative expression of BGP mRNA of osteoblasts tended to decrease quadratically(P<0.10). This suggested that suitable dose of vitamin A up-regulated the relative expression of the ALP mRNA and BGP mRNA of the osteoblasts, and high levels of vitamin A down-regulated its expression.
⑥The present results implied that the mechanisms for vitamin A excess to affect calcium and phosphorous deposition in bone of broiler chickens were probably that the excessive vitamin A depressed the relative expressions of the ALP mRNA and BGP mRNA in tissues and osteoblasts, which lowered the synthesis of the ALP and BGP and resulted in the negative effects to the despositions of the calcium and phosphorous in bone.
在本研究的试验条件下初步得出以下结果:
①肉鸡胫骨钙、磷沉积和胫骨强度与日粮维生素A水平呈显著或趋于显著的剂量依赖关系(P<0.05;P<0.10)。其中,胫骨钙磷沉积与胫骨强度以3000~6000 IU/kg维生素A组较高;30000~60000 IU/kg维生素A组较低;日粮维生素A水平为15000 IU/kg时,胫骨钙、磷沉积与胫骨强度也呈一定降低趋势。
②日粮维生素A水平在3000~60000 IU/kg范围内,日粮维生素A水平以剂量依赖模式影响肉鸡的血清PTH、CT、1,25(OH)_2D_3、BGP与PINP浓度及血清ALP和BAP活性(P<0.05;P<0.10)。其中,30000~60000 IU/kg的高剂量维生素A引起肉鸡血清中PTH浓度升高,CT与1,25(OH)2D3浓度及BGP与PINP浓度降低,ALP与BAP活性降低。
③随日粮维生素A添加剂量的升高,肉鸡胫骨、肝脏及小肠组织中ALP mRNA和BGP mRNA相对表达量均呈不同程度降低,二者呈显著(P<0.05)或趋于显著(P<0.10)的一次线性或二次曲线剂量依赖关系;其表达量均以3000~6000 IU/kg维生素A日粮组较高,30000~60000 IU/kg维生素A日粮组较低,15000IU/kg维生素A日粮组居中。提示日粮维生素A过量可下调肉鸡胫骨和肝脏及小肠组织中ALP mRNA和BGP mRNA的相对表达量。
④随培养液中维生素A水平的增加,肉鸡成骨细胞培养液中ALP活性、BGP、PINP、OPN和OPG浓度呈显著的一次线性降低(P<0.05)或二次曲线降低趋势(P<0.10)。提示维生素A水平以剂量依赖模式影响肉鸡成骨细胞的功能和活性,适宜的维生素A水平可促进成骨细胞的活性,诱导其分化成熟,但高剂量的维生素A抑制成骨细胞的活性和分化成熟。
⑤随培养液中维生素A水平的增加,成骨细胞ALP mRNA相对表达量呈显著的一次线性或一元二次降低(P<0.05),BGP mRNA相对表达量呈一元二次降低趋势(P<0.10)。提示维生素A以剂量依赖模式影响成骨细胞ALP mRNA与BGP mRNA相对表达量,适宜剂量维生素A可上调其表达,高剂量维生素A则下调其表达,低剂量维生素A对BGP mRNA相对表达量也有一定抑制效果。
⑥综合试验1~4的结果,维生素A过量影响肉鸡钙磷沉积的分子机理可能是:过量维生素A抑制了组织和成骨细胞ALP mRNA和BGP mRNA的相对表达量,降低了ALP和BGP的合成,进而对骨骼代谢及钙、磷沉积造成负面影响。
The present study was conducted to investigate the effects of vitamin A excess on calcium and phosphorus deposition and alkaline phosphatase and bone gla-protein mRNA expression in broiler chickens. The first three experiments were conducted by using in vivo experimental techniques. A total of 450 one-day-old Arbor Acre half male and half female broiler chickens with similar body weight were randomly allotted to five dietary treatments with six replicate cages per dietary treatment and fifteen birds per cage. The basal diet that didn’t contain vitamin A was mainly composed of corn and soybean meal.The broiler chickens in the five treatments were fed the basal diets supplemented with 3000, 6000, 15000, 30000 or 60000 IU/kg vitamin A in a single factorial arrangement. The trial lasted for 42 days. The objective of the experiment 1 was to elucidate the effects of vitamin A excess on contents of Ca, P in serum and concentrations of ash, Ca and P and BMD in tibia of broilers, and further to demonstrate the negative effects of excessive vitamin A on calcium and phosphorus deposition in broiler chickens. The experiment 2 was conducted to investigate the effects of vitamin A excess on hormones related to calcium and phosphorus deposition and biochemical markers of bone formation. The experiment 3 was conducted to study the effects of vitamin A excess on relative expression of ALP mRNA and BGP mRNA in small intestine, tibia and liver, and to elucidate the molecular mechanisms for vitamin A excess to affect calcium and phosphorus deposition in broiler chickens. The experiment 4 was conducted to demonstrate the effects of different levels of vitamin A(0, 0.03, 0.10, 0.30, 1.00, 2.00, 6.00, 12.00, 24.00μg/ml) on osteoblasts in vitro by evaluating cells activity and relative expression of ALP mRNA and BGP mRNA of osteoblastic cells and further to investigate the molecular mechanisms by which vitamin A modulated calcium and phosphorus deposition.
Under the present experimental conditions, the results showed as follows:
①Dietary vitamin A influenced the strength and disposition of calcium and phosphours in the tibia of broilers in a dose-dependent manner(P<0.05;P<0.10). The supplementation of 3000~6000 IU/kg vitamin A in the diet improved the strength and disposition of calcium in the tibia. But the strength and disposition of calcium in the tibia tended to be suppressed when the addition of vitamin A in diet was increased to 30000~60000 IU/kg. And the tibia strength tended to be lower while 15000 IU/kg vitamin A was added in the diet.
②Dietary vitamin A influenced contents of PTH, CT, 1,25(OH)2D3, BGP and PINP as well as ALP and BAP activity in serum in a dose-dependent manner (P<0.05; P<0.10) with the supplementation of vitamin A in the diet from 3000 to 60000 IU/kg. The addition of 30000～60000 IU/kg vitamin A to the diet increased serum PTH level, and depressed the contents of CT, 1,25(OH)2D3, BGP and PINP as well as ALP and BAP activity in serum of broilers.
③The dietary vitamin A influenced the relative expressions of the ALP mRNA and BGP mRNA in the tibia, liver and small intestine in a linear or quadratical dose-dependent manner (P<0.05, P<0.10). The addition of 3 000~6 000 IU/kg vitamin A to the diet showed the better effect in relative expressions of ALP mRNA and BGP mRNA in tibia, liver and the small intestine of broilers. However, the addition of 3 0000~6 0000 IU/kg vitamin A to the diet could down-regulate the relative expressions of ALP mRNA and BGP mRNA, and the addition of 15 000 IU/kg vitamin A tended to decrease in relative expressions of ALP mRNA and BGP mRNA.
④With the increase of vitamin A in the culture, ALP activity and concentrations of BGP, PINP, OPN and OPG in the culture of the cranium osteoblasts of broilers decreased linearly or quadratically(P<0.05, P<0.10). This suggested that the vitamin A could promote the secretion functions and activity of cranium osteoblasts in broiler chickens in a dose-dependent manner. Adequate doses of vitamin A could promote the activity of osteoblasts, and induce differentiation and maturation. But the large doses of vitamin A was less effective.
⑤With the increase of vitamin A in the culture of the osteoblasts in broilers, the relative expression of the ALP mRNA of osteoblasts decreased linearly or quadratically(P<0.05), and the relative expression of BGP mRNA of osteoblasts tended to decrease quadratically(P<0.10). This suggested that suitable dose of vitamin A up-regulated the relative expression of the ALP mRNA and BGP mRNA of the osteoblasts, and high levels of vitamin A down-regulated its expression.
⑥The present results implied that the mechanisms for vitamin A excess to affect calcium and phosphorous deposition in bone of broiler chickens were probably that the excessive vitamin A depressed the relative expressions of the ALP mRNA and BGP mRNA in tissues and osteoblasts, which lowered the synthesis of the ALP and BGP and resulted in the negative effects to the despositions of the calcium and phosphorous in bone.
引文
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