脂联素对围产期奶牛脂肪动员的相关性研究
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摘要
本文通过体外实验、健康奶牛和自然发病奶牛实验,应用荧光定量RT-PCR方法,观测了体外添加神经内分泌因子和代谢中间产物时,以及奶牛围产期间、酮病牛的脂肪细胞因子脂联素(ADPN)与脂肪动员关键酶基因(HSL)表达的规律,同时对健康奶牛和酮病牛的血液相关指标进行了监测。
试验结果表明:一定范围内的胰岛素,抑制HSL mRNA的表达并呈剂量依赖性,高浓度时对ADPN mRNA表达呈抑制作用且有剂量依赖性;高浓度的胰高血糖素促进HSL mRNA和ADPN mRNA的表达;低浓度的NEFA对ADPN mRNA表达具有促进作用,高浓度时抑制ADPN mRNA的表达;低浓度NEFA对HSL mRNA的表达有抑制作用,呈剂量依赖性,但在高浓度时反而促进了其表达;高浓度的BHBA显著下调HSL mRNA和ADPN mRNA的表达;高浓度的GLU抑制ADPN mRNA的表达,对HSL mRNA表达具有促进作用。代谢中间产物通过促进ADPN mRNA或抑制HSL mRNA的表达来调节脂肪代谢。
在健康奶牛的围产期,脂肪组织ADPN mRNA和HSL mRNA变动趋势相同且呈正相关,与机体能量代谢状态一致。初步推测ADPN是通过调控HSL mRNA的表达和分泌来调节围产期奶牛脂肪动员的。
酮病牛血糖显著降低,血浆NEFA和BHBA明显增高,血清INS降低和INS/GN变小,脂肪组织HSL mRNA的表达低于产后健康奶牛,但ADPN mRNA却极显著地高于健康奶牛产后各时段。酮病牛脂肪组织ADPN mRNA表达的上调和HSL mRNA表达下调可能与脂肪动员代谢产物NEFA和BHBA的增加有关。
生理性和病理性能量负平衡对HSL mRNA与ADPN mRNA表达的影响不同,其原因可能与血糖降低的程度和脂肪动员的代谢产物NEFA和BHBA增加的程度的不同有关。
In this study, healthy multiparous dairy cows, the diseased dairy cows with ketosis, in vitro primary cell culture, and quantitive RT-PCR were applied to research the expressing level of ADPN and HSL, and their relation. The focal point of this study is to probe the regulation mechanism of ADPN on fat mobilization.
First of all, according to the publishing sequence, four pairs of primers were designed and RT-PCR was employed to amplify the partial cDNA of HSL and ADPN, and four target sequences were recombinated into cloning vector respectively, then homology of the two long sequence plasmids was 99.6% and 85% for 299bp fragment of HSL and 480bp fragment of ADPN respectively, the two short sequences were 100% and 96.7% for 91bp fragment of HSL and 99bp fragment of ADPN respectively. The method of fluorescent quantitation RT-PCR was established to detect ADPN mRNA and HSL mRNA of adipose tissue.
Then, bovine preadipocyte was cultured by the method of adipocyte culture reported by previous literature. The effect of bovine insulin (0、5、10、20、50、100nmol/L)and bovine glucagon(0、25、100、200、500、1000pg/ml)on ADPN mRNA and HSL mRNA were detected by fluorescent quantitation PCR.The result showed that the expression level of HSL mRNA in adipocyte gradually decreased with increasing concentration of INS in culture media from 5nmol/L to 100nm/L. When up to 20 nmol/L, depression effect became strengthen with dose dependent. The expression level of HSL mRNA in adipocyte gradually increased with increasing concentration of Glucagon in culture media from 25pg/mL to 1000pg/mL. When over 25pg/mL, it appearded with dose dependent. The inhibited effect of INS on the expression of ADPN showed dose dependent with the increasing. Glucagon promote the expression level of ADPN mRNA, but over 500pg/L, had significant effect(p<0.01). There is a positive relation between ADPN mRNA and HSL mRNA。
The effect of NEFA (0.2、0.4、0.8、1.6、3.2 nmol/l),β-hydroxybutyric acid(0.3、0.6、1.2、2.4、4.8 nmol/l) and glucose(0、5、10、15、20、25 mmol/l)on ADPN mRNA and HSL mRNA were detected by fluorescent quantitation PCR also. the results indicated that:①the expression of ADPN mRNA were promoted by the NEFA from 0.2 nmol/l to 0.8 nmol/l with dose dependent。But the expression of ADPN mRNA were Down-regulated by the high concentration of NEFA(﹥1.6 nmol/l).and the effect of NEFA on the expression of HSL mRNA was opposite with ADPN.②the lower concentration of BHBA had no significant effect on the expression of ADPN mRNA and HSL mRNA. The higher concentration of BHBA significant down-regulated the levels of ADPN mRNA and HSL mRNA.③5 nmol/l Glucose down-regulated ADPN mRNA remarkably, but dose dependent was not observed. High density glucose(20mmol/L、25mmol/L)promoted the expression of HSL mRNA, while the low desity group did not.The above results show that : intermediate metabolic products regulate the fat metabolism through promoting the expression ADPN mRNA or depressing the expression of HSL mRNA.In the experiment of healthy periparturent cows , Ten cows (age, 5-8years old ;parity number,3-5;mean milk yield in a 305 lactation 5000Kg) before to day 28 after parturition. Cows were fed on a total mixed ration (TMR) ad libitum thrice daily. Nutrient composition and level of TMR varied with the stage of pregnancy and lactation. Fatty tissue500-1000mg are collected by thin needle sucking technique from tail subcutaneous in antepartum28d,14d,and post partum1d,14d,28d。Copy number of ADPN mRNA and HSL mRNA in adipose tissue were determined by fluorescent quantitation PCR.Abundance of ADPN mRNA decreased with near parturition, and reached the minimum at 1d after parturition and then increased gradually ,but was still lower at 28d after parturition than that of 28d before parturition ,HSL mRNA level in fatty tissue was expressed before parturition,and reached the minimum value at 1d after parturition and then increased gradually ; Changes of ADPN mRNA as HSL mRNA was identical with the condition of energy metabolism .Adiponectin could adjust fat mobilization through regulating the expression and secretion of HSL mRNA.
In the expeiment of ketotic cows,the tail fat tissue were collected from 7 cows with ketosis and 7 peripartum cows at 1d, 14d and 28d postpartum, and then expression level of ADPN mRNA and HSL mRNA were determined by fluorescent quantitation PCR.Contents of GLUTG、FFA、INS、GN and Leptin in blood collected from cows with ketosis and healthy cows were determined. The results were as follows:①cows with ketosis displayed hypoglycemia,increase of contents of serum NEFA and BHBA and decrease of contents of serum INS.②HSL mRNA expression level was lower in cows with ketosis than that of postpartum cows, but ADPN mRNA expression level of cows with ketosis was higher than postpartum cows indicating that different changes of ADPNmRNA and HSLmRNA in fat tissue from ketotic and health cows may be related to increasing concentration of blood Nefa and BHBA,and the serious degree of negative energy balance of periparturent cows
试验结果表明:一定范围内的胰岛素,抑制HSL mRNA的表达并呈剂量依赖性,高浓度时对ADPN mRNA表达呈抑制作用且有剂量依赖性;高浓度的胰高血糖素促进HSL mRNA和ADPN mRNA的表达;低浓度的NEFA对ADPN mRNA表达具有促进作用,高浓度时抑制ADPN mRNA的表达;低浓度NEFA对HSL mRNA的表达有抑制作用,呈剂量依赖性,但在高浓度时反而促进了其表达;高浓度的BHBA显著下调HSL mRNA和ADPN mRNA的表达;高浓度的GLU抑制ADPN mRNA的表达,对HSL mRNA表达具有促进作用。代谢中间产物通过促进ADPN mRNA或抑制HSL mRNA的表达来调节脂肪代谢。
在健康奶牛的围产期,脂肪组织ADPN mRNA和HSL mRNA变动趋势相同且呈正相关,与机体能量代谢状态一致。初步推测ADPN是通过调控HSL mRNA的表达和分泌来调节围产期奶牛脂肪动员的。
酮病牛血糖显著降低,血浆NEFA和BHBA明显增高,血清INS降低和INS/GN变小,脂肪组织HSL mRNA的表达低于产后健康奶牛,但ADPN mRNA却极显著地高于健康奶牛产后各时段。酮病牛脂肪组织ADPN mRNA表达的上调和HSL mRNA表达下调可能与脂肪动员代谢产物NEFA和BHBA的增加有关。
生理性和病理性能量负平衡对HSL mRNA与ADPN mRNA表达的影响不同,其原因可能与血糖降低的程度和脂肪动员的代谢产物NEFA和BHBA增加的程度的不同有关。
In this study, healthy multiparous dairy cows, the diseased dairy cows with ketosis, in vitro primary cell culture, and quantitive RT-PCR were applied to research the expressing level of ADPN and HSL, and their relation. The focal point of this study is to probe the regulation mechanism of ADPN on fat mobilization.
First of all, according to the publishing sequence, four pairs of primers were designed and RT-PCR was employed to amplify the partial cDNA of HSL and ADPN, and four target sequences were recombinated into cloning vector respectively, then homology of the two long sequence plasmids was 99.6% and 85% for 299bp fragment of HSL and 480bp fragment of ADPN respectively, the two short sequences were 100% and 96.7% for 91bp fragment of HSL and 99bp fragment of ADPN respectively. The method of fluorescent quantitation RT-PCR was established to detect ADPN mRNA and HSL mRNA of adipose tissue.
Then, bovine preadipocyte was cultured by the method of adipocyte culture reported by previous literature. The effect of bovine insulin (0、5、10、20、50、100nmol/L)and bovine glucagon(0、25、100、200、500、1000pg/ml)on ADPN mRNA and HSL mRNA were detected by fluorescent quantitation PCR.The result showed that the expression level of HSL mRNA in adipocyte gradually decreased with increasing concentration of INS in culture media from 5nmol/L to 100nm/L. When up to 20 nmol/L, depression effect became strengthen with dose dependent. The expression level of HSL mRNA in adipocyte gradually increased with increasing concentration of Glucagon in culture media from 25pg/mL to 1000pg/mL. When over 25pg/mL, it appearded with dose dependent. The inhibited effect of INS on the expression of ADPN showed dose dependent with the increasing. Glucagon promote the expression level of ADPN mRNA, but over 500pg/L, had significant effect(p<0.01). There is a positive relation between ADPN mRNA and HSL mRNA。
The effect of NEFA (0.2、0.4、0.8、1.6、3.2 nmol/l),β-hydroxybutyric acid(0.3、0.6、1.2、2.4、4.8 nmol/l) and glucose(0、5、10、15、20、25 mmol/l)on ADPN mRNA and HSL mRNA were detected by fluorescent quantitation PCR also. the results indicated that:①the expression of ADPN mRNA were promoted by the NEFA from 0.2 nmol/l to 0.8 nmol/l with dose dependent。But the expression of ADPN mRNA were Down-regulated by the high concentration of NEFA(﹥1.6 nmol/l).and the effect of NEFA on the expression of HSL mRNA was opposite with ADPN.②the lower concentration of BHBA had no significant effect on the expression of ADPN mRNA and HSL mRNA. The higher concentration of BHBA significant down-regulated the levels of ADPN mRNA and HSL mRNA.③5 nmol/l Glucose down-regulated ADPN mRNA remarkably, but dose dependent was not observed. High density glucose(20mmol/L、25mmol/L)promoted the expression of HSL mRNA, while the low desity group did not.The above results show that : intermediate metabolic products regulate the fat metabolism through promoting the expression ADPN mRNA or depressing the expression of HSL mRNA.In the experiment of healthy periparturent cows , Ten cows (age, 5-8years old ;parity number,3-5;mean milk yield in a 305 lactation 5000Kg) before to day 28 after parturition. Cows were fed on a total mixed ration (TMR) ad libitum thrice daily. Nutrient composition and level of TMR varied with the stage of pregnancy and lactation. Fatty tissue500-1000mg are collected by thin needle sucking technique from tail subcutaneous in antepartum28d,14d,and post partum1d,14d,28d。Copy number of ADPN mRNA and HSL mRNA in adipose tissue were determined by fluorescent quantitation PCR.Abundance of ADPN mRNA decreased with near parturition, and reached the minimum at 1d after parturition and then increased gradually ,but was still lower at 28d after parturition than that of 28d before parturition ,HSL mRNA level in fatty tissue was expressed before parturition,and reached the minimum value at 1d after parturition and then increased gradually ; Changes of ADPN mRNA as HSL mRNA was identical with the condition of energy metabolism .Adiponectin could adjust fat mobilization through regulating the expression and secretion of HSL mRNA.
In the expeiment of ketotic cows,the tail fat tissue were collected from 7 cows with ketosis and 7 peripartum cows at 1d, 14d and 28d postpartum, and then expression level of ADPN mRNA and HSL mRNA were determined by fluorescent quantitation PCR.Contents of GLUTG、FFA、INS、GN and Leptin in blood collected from cows with ketosis and healthy cows were determined. The results were as follows:①cows with ketosis displayed hypoglycemia,increase of contents of serum NEFA and BHBA and decrease of contents of serum INS.②HSL mRNA expression level was lower in cows with ketosis than that of postpartum cows, but ADPN mRNA expression level of cows with ketosis was higher than postpartum cows indicating that different changes of ADPNmRNA and HSLmRNA in fat tissue from ketotic and health cows may be related to increasing concentration of blood Nefa and BHBA,and the serious degree of negative energy balance of periparturent cows
引文
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