【作者】 高静；

【导师】 林海；

【作者基本信息】 山东农业大学 ， 动物营养与饲料科学， 2011， 硕士

【摘要】 为探讨糖皮质激素对肉仔鸡骨骼肌蛋白代谢的影响,本研究从体内试验和体外试验两方面入手,体内试验用人为导入外源糖皮质激素的方法,体外试验离体培养骨骼肌成肌细胞并用糖皮质激素处理,观察糖皮质激素对蛋白的合成途径和蛋白的分解途径的数个关键基因mRNA的相对表达量的变化情况,为骨骼肌蛋白代谢的基因表达调控的网络构建以及研究骨骼肌蛋白代谢的详细的分子机制提供理论依据。体内试验分两部分:试验一选取28日龄体重相近的AA雄性肉鸡288只,随机分为正常日粮组和高蛋白日粮组,经过三天日粮适应期后,每个组随机分为三个处理:糖皮质激素处理组(DEX)、对照组、限饲组,每个处理3个重复,每个重复12只鸡,糖皮质激素处理组8:30和20:30,腹部皮下注射地塞米松1.0mg/kg体重,对照组、限饲组注射与糖皮质激素处理组等体积的生理盐水,限饲组饲喂糖皮质激素处理组前一天的采食量。34日龄晚20:30开始禁食。禁食后再饲喂组在35日龄早上8:30再饲喂一小时后采样。各试验鸡均翅静脉采血,3000r/min离心10min,分离血浆,-20℃冷冻保存。取胸大肌(M. pectoralis major,PM),液氮速冻,-80℃贮存。同时取部分组织样品。取全部胸肌称重,测定胸肌指数(胸肌重/活体重),取右腿全部肌肉称重,测定腿肌指数(腿肌重/活体重)。结果表明,糖皮质激素能使日增重显著降低,采食量无显著影响。糖皮质激素能使血浆中尿酸、T-AA、胰岛素的浓度显著上升。糖皮质激素能显著上调蛋白质分解途径相关基因的表达,对蛋白质合成途径的相关基因的抑制作用不明显。高蛋白日粮对缓解糖皮质激素的作用不明显。试验二选取35日龄体重相近的AA雄性肉鸡72只,随机分为2个组:糖皮质激素处理组(DEX)和对照组,每个处理3个重复,每个重复6只鸡。36-38日龄,8:00和20:00,糖皮质激素处理组皮下注射地塞米松1.0mg/kg体重,对照组注射相同体积的生理盐水。38日龄20:00禁食12h,禁食后(每个处理3个重复,每个重复取4只鸡)空腹采血,然后每个处理分A、B两个处理进行灌服处理:A组灌服亮氨酸500mg/Kg体重,B组灌服生理盐水5ml,灌服后1h,各试验鸡均翅静脉采血,3000r/min离心10min,分离血浆,-20℃冷冻保存。取胸大肌(M. pectoralis major,PM),液氮速冻,-80℃贮存。同时取部分组织样品。取全部胸肌称重,测定胸肌指数(胸肌重/活体重),取右腿全部肌肉称重,测定腿肌指数(腿肌重/活体重)。结果表明,糖皮质激素能使日增重显著降低,采食量无显著影响。糖皮质激素能显著促进蛋白质分解途径相关基因的表达。灌服对缓解糖皮质激素的作用也不明显。体外试验分两个试验:试验三培养骨骼肌成肌细胞,细胞在含有10%的胎牛血清的DMEM培养基中生长,当细胞铺满六孔板的70%时,更换成含有2%马血清的DMEM培养基诱导成肌细胞分化成肌管,分化时间为48h,分化成肌管的成肌细胞经过1h的无血清处理后,用处理液处理,试验分四个处理:正常培养基、地塞米松处理(1um)、胰岛素处理(100nm)、地塞米松和胰岛素共同处理(DEX:1um;INS:100nm),处理液处理3h。结果表明,糖皮质激素能显著抑制IGF-I mRNA的表达,显著上调MyoG mRNA的表达,INS能显著上调MyoG mRNA的表达,显著抑制蛋白质分解途径的相关基因的表达,在INS存在时,糖皮质激素能上调MyoD、Atrogin-1的表达,提示糖皮质激素参与了细胞的新陈代谢。试验四是使用已分化成肌管的成肌细胞经过1h的无血清处理后,再用处理液处理,试验分两个处理:正常培养基、RU486(100nm)和地塞米松(1um)共同处理,正常培养基处理4h,RU486处理1h后,再用RU486和DEX共同处理3h。结果表明,RU486对IGF-I、MyoD、MSTN、MuRF1mRNA的表达量均无显著影响,说明RU486能抑制DEX的作用。更多还原

【Abstract】 In order to study the effects of glucocorticoids on the protein metabolism of skeletal muscles in broiler chickens (Gallus gallus domesticus), we did the experiments in vivo and in vitro. In vivo, we used exogenous glucocorticoids to induce stress on the later stage of broilers’development, and in vitro, we treated the myoblasts of broiler chickens with dexamethasone. The mRNA level of several genes which were important for the protein metabolism were measured looking forward to constructing genetic regulatory networks of skeletal muscle protein metabolism as well as finding out its’detailed molecular mechanism.The experiments in vivo were separated into two parts. In trial 1, 144 male AA broilers of 28 day were allocated into 12 treatments according to body weigh, 12 broilers per treatment. At the age of 29d, chickens were randomly separated into two groups: the basal diet group and the high protein ration, of which the latter was derived from a basal diet group through a 3d adjustment. At the age of 32d, chickens were randomly subjected to one of the following three treatments for 3 days: subcutaneous injection of DEX (2mg/kg BM), sham-treated (2 mg/kg BM of saline, control), sham-treated and limited-fed treatment to keep the feed consumption as that of the DEX chickens in the previous day. BM was recorded daily. At the age of 35d, half chickens of each treatment were randomly exposed to fasting for 12h or fasting 12h and refeeding 1h before samples were obtained. A blood sample was drawn from a wing vein by using a heparinized syringe within 30s and collected in iced tubes. Plasma was obtained after centrifugation at 400g for 10 min at 4°C and was stored at ?20°C for further analysis. Immediately after the blood sample had been obtained, the chicks were selected and sacrificed by exsanguination, and then harvested and weighed the breast and thigh muscle respectively. A 1 to 2g sample was respectively obtained from left PM, cooled down in liquid nitrogen and stored at -80℃for further analysis. The muscle of right leg and whole breast were harvested and weighed respectively, the breast and thigh mass were expressed as percentage of body mass (%). After three days’treatment, DEX had a significantly decrease (P<0.01) on daily gain of broiler chickens, in contrast, DEX had no significant (P>0.05) effect on food intake. The concentration of plasma uric acid (UA), total amino acid (T-AA) and insulin (INS) were higher at both sampling ages. The mRNA level of several genes that are important for protein proteolysis were significantly higher after three days exposure. The effect of glucocorticoid was not significantly inhibited by the high protein ration. In trial 2, 72 male AA broilers of 35 day were allocated into 12 treatments according to body weigh, 6 broilers per treatment. At the age of 36d, chickens were randomly subjected to one of the following two treatments for 3 days: subcutaneous injection of DEX (2mg/kg BM), sham-treated (2mg/kg BM of saline, control). BM was recorded daily. At the age of 38d, chickens of each treatment were exposed to fasting for 12h before blood samples were obtained. A blood sample was drawn from a wing vein by using a heparinized syringe within 30s and collected in iced tubes. After obtained the blood samples, every treatment was randomly separated into two perfusing treatments, leucine (500mg/kg BM) and salt (5ml). After perfusing 1h, another blood sample was drawn from a wing vein by using a heparinized syringe within 30 s and collected in iced tubes. Plasma was obtained after centrifugation at 400g for 10 min at 4°C and was stored at ?20°C for further analysis. Immediately after the blood sample had been obtained, the chicks were selected and sacrificed by exsanguination, and then harvested and weighed the breast and thigh muscle respectively. A 1 to 2g sample was respectively obtained from left PM, cooled down in liquid nitrogen and stored at -80℃for further analysis. The muscle of right leg and whole breast were harvested and weighed respectively, the breast and thigh mass were expressed as percentage of body mass (%). After three days treatment, DEX had a significantly decrease (P<0.01) on daily gain of broiler chickens, in contrast, DEX had no significant effect on food intake. The mRNA level of several genes that are important for protein proteolysis were significantly higher after three days exposure, while the mRNA level of genes that are important for protein synthesis were not significantly affected. The effect of glucocorticoid was not significantly inhibited by the perfusing of leucine. The experiments in vitro were separated into two parts also. In trial 3, cells were cultured in DMEM medium containing 10% heat-inactivated FBS, when cells were 70% confluent, the proliferation medium was replaced by a differentiation medium containing 2% horse serum. After 48h of differentiation, cells were incubated for 1h with serum-free DMEM and subjected to different treatments for three hours including serum-free DMEM, 1uM Dexamethasone, 100nM Insulin, 1uM Dexamethasone and 100nM Insulin, four random fields of each of the six replicates were selected. The mRNA level of IGF-I was significantly inhibited by the treatment of dexamethasone after three hours’treatment, and the mRNA level of MyoG was significantly increased by the treatment of dexamethasone. The mRNA level of MyoG was significantly increased by the treatment of insulin, and the mRNA levels of several genes that are important for protein proteolysis were significantly inhibited by the treatment of insulin after three hours’treatment. When insulin was in the medium, the mRNA level of MyoD and Atrogin-1 was significantly increased by the treatment of glucocorticoid, which indicated that glucocorticoids had taken part in the metabolism of myoblasts. In trial 4, after 48h of differentiation, cells were incubated for 1h with serum-free DMEM and subjected to different treatments for three hours including serum-free DMEM, 100nm and RU486 1uM Dexamethasone (after the treatment of RU486 alone for 1h, the cells were treated with RU486 and dexamethosone for 3h), two random fields of each of the six replicates were selected. The mRNA levels of IGF-I、MyoD、MSTN、MuRF1 were not significantly affected by the treatment, which indicated that the effects of dexamethasone could be inhibited by the RU486.更多还原