不同浓度氨基酸对仔猪原代肝细胞尿素循环的影响

Effects of different concentrations of amino acids on urea cycle in primary porcine hepatocytes

【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。

【Objective】To investigate the effects of different concentrations of amino acids on urea cycle in primary hepatocytes isolated from piglet, and explore the potential mechanism.【Method】Primary hepatocytes isolated from5-day old piglet were cultured in medium containing different concentrations of amino acids(AA)(1, 2, or 4 times of physiological concentration in serum). After 24 h, the supernatant and cells were collected. Urea and ammonia concentrations in the supernatant, glutaminase(GLS) and glutamine synthetase(GS) activities and ammonia concentration in cells were examined by colorimetry. The m RNA expression of genes involved in urea cycle,including carbamyl phosphate synthetase 1(CPS1), Argininosuccinate synthetase(ASS), Argininosuccinate lyase(ASL),Omithine transcarbamylase(OTC), Arginase(ARG) were detected by q RT-PCR.【Result】Urea and ammonia concentrations in the supernatant, and GLS activity in cells were significantly elevated in 4×AA group. Meanwhile,4×AA remarkably increased the m RNA expression of CPS1, ASS and ASL genes in hepatocytes. NH4 Cl at 1.0 and2.0 mmol·L^(-1) concentrations significantly promoted urea synthesis in the cell.【Conclusion】High concentration of AA might accelerate ammonia accumulation by GLS and enhance the expression of the urea cycle enzymes(CPS1,ASS and ASL), which contribute to increased production of urea.