不同浓度氨基酸对仔猪原代肝细胞尿素循环的影响

发布时间：2018-05-27 00:39

本文选题：仔猪 + 原代肝细胞　；参考：《华南农业大学学报》2017年05期

【摘要】：【目的】研究不同浓度的氨基酸对仔猪原代肝细胞尿素循环的影响及其机理。【方法】5日龄仔猪门静脉灌流后从中分离纯化出肝细胞并进行培养,在培养基中添加不同浓度的氨基酸(分别为猪血液中氨基酸生理浓度的1、2和4倍),24 h后收集上清和细胞。用比色法检测上清中尿素、底物氨和细胞中氨的浓度以及谷氨酰胺酶(Glutaminase,GLS)和谷氨酰胺合成酶(Glutamine synthetase,GS)的活性,实时荧光定量PCR法检测细胞中氨甲酰磷酸合成酶1(Carbamyl phosphate synthetase 1,CPS1)、精氨基琥珀酸合成酶(Argininosuccinate synthetase,ASS)、精氨琥珀酸裂解酶(Argininosuccinate lyase,ASL)、鸟氨酸氨甲酰转移酶(Omithine transcarbamylase,OTC)和精氨酸酶(Arginase,ARG)等尿素循环相关酶基因m RNA的表达。【结果】4倍氨基酸组显著提高了仔猪原代肝细胞培养上清中氨和尿素的浓度,增强了细胞中GLS的活性。荧光定量PCR结果表明,4倍氨基酸组显著提高了细胞中CPS1、ASS和ASL等基因m RNA的表达。1.0和2.0 mmol·L~(-1)的NH4Cl显著促进细胞尿素的合成。【结论】在仔猪原代肝细胞中,高浓度氨基酸可以通过提高GLS的活性等途径加速氨的积累,促进CPS1、ASS、ASL等尿素循环相关酶基因m RNA的表达,从而促进尿素的生成。
[Abstract]:[objective] to study the effect of different concentrations of amino acids on urea circulation in primary hepatocytes of piglets and its mechanism. [methods] Hepatocytes were isolated and cultured from 5-day-old piglets after portal vein perfusion. Supernatants and cells were collected by adding different concentrations of amino acids (1, 2 and 4 times of physiological concentration of amino acids in pig blood) to the culture medium for 24 h. The concentrations of urea, substrate ammonia and ammonia in the supernatant, and the activities of glutamine synthetase Glutaminase (GLSs) and glutamine synthetase (Glutamine synthetaseGlutamine) were determined by colorimetric method. Real-time fluorescence quantitative PCR assay for the detection of urea cycle related to carbamyl phosphate synthase (1(Carbamyl phosphate synthetase 1), argino succinate synthetase (Argino succinate synthetase), arginino succinate lyase (Arginino succinate lyase), ornithine carbamyltransferase (Omithine transcarbamyltransferase) and arginase (ARG) in cells [results] 4-fold amino acid group significantly increased the concentration of ammonia and urea in the supernatant of primary hepatocyte culture of piglets. The activity of GLS was enhanced. The results of fluorescence quantitative PCR showed that 4 fold amino acid group significantly increased the expression of m RNA, such as CPS1, ASS and ASL, and the NH4Cl of 2. 0 and 2. 0 mmol Ln-1) significantly promoted the synthesis of urea. [conclusion] in the primary hepatocytes of piglets, the expression of CPS1, ASS and ASL genes was significantly increased. The high concentration of amino acids can accelerate the accumulation of ammonia by increasing the activity of GLS, and promote the expression of the gene m RNA of urea cycle related enzymes such as CPS1, ASS, ASL and so on, thus promoting the formation of urea.
【作者单位】：华南农业大学动物科学学院;
【基金】：973项目(2013CB127304)
【分类号】：S828

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