严重创伤对肠上皮细胞中性氨基酸载体ASCT2表达的影响

The influence of serious trauma on the expression of neutral amino acid transporter ASCT2 in human intestinal epithelial cells

目的：研究用肠上皮细胞系Caco-2定量分析在严重创伤情况下氨基酸的转运变化及其载体蛋白表达情况。方法：体外培养肠上皮细胞系Caco-2,首先分析其细胞膜表面Na^＋-依赖性中性氨基酸转运及其转运载体功能特性。将细胞置于缺氧、营养剥夺和缺血等创伤条件下,继续培养1-6 h,观察其刷状缘膜囊氨基酸转运及其相应转运载体蛋白及mRNA表达情况。结果：Caco-2细胞膜表面Na^＋-依赖性L-丙氨酸的转运速率,高于L-谷氨酰胺（876±67.5）pmol/mg蛋白·min^-1vs（635±52.8）pmol/mg蛋白·min^-1,P〈0.01）。在主要的中性氨基酸载体中,仅仅成功扩增出ASCT2的mRNA条带。与对照组相比,在营养剥夺及缺血条件下,L-丙氨酸和L-谷氨酰胺的转运速率明显下降（P〈0.01）,而缺氧对氨基酸转运无明显影响。这种变化主要引起转运动力学Vmax值下降,Km值无明显变化。与对照组相比,营养剥夺及缺血条件下相关转运载体蛋白及mRNA表达下降。结论：严重创伤对Na^＋-依赖性中性氨基酸转运及关键性转运载体调控特点不同。这对临床上危重症病人特殊的营养底物需求,可能提供积极的理论依据。

Objective： Serious trauma to the body is often associated with changes in protein metabolism of multiple organs and tissues. Clinically,the catabolic response results in a generalized negative nitrogen balance.Nutrition support has been an important component of the care of seriously traumatized patients.Of this,preventing intestinal mucosal atrophy,maintaining immunocompetence and preserving gut flora,total enteral nutrition（TEN） has been the primary mode of nutrition support.However,during states of severe trauma,enterocyte transport function remains unclear.This study aims to quantitate the Na^＋-dependent neutral amino acid transport and expression of its transporter in traumatically injured Caco-2 cell lines.Methods： Transport and transporter of Na^＋-dependent neutral amino acid in Caco-2 cell lines were characterized.Then the cell lines were cultured under hypoxic,nutrient-deprived and ischemic conditions for 1-6h.After severe trauma was performed,we investigated the transport of Na^＋-dependent neutral amino acids and the expression of transporter protein and mRNA in apical membrane vehicles.Results： The transport velocity of Na^＋-dependent L-alanine was faster than that of L-glutamine [（876±67.5） pmol/mg protein·min^-1 vs（635±52.8） pmol/mg protein·min^-1,（P〈0.01）].Among the neutral amino acid transporters,only ASCT2 mRNA was successfully amplificated.Under nutrient-deprived and ischemic conditions,transport of L-alanine and L-glutamine decreased significantly compared with control（P〈0.01）,whereas hypoxia taked no significant effect.The changes were associated with a decrease in Vmax without an affect on Km.Expression of relative transporter proteins and mRNA decreased significantly compared with control（P〈0.01）.Conclusion： Na^＋-dependent neutral amino acid transport and its key transporter may be differentially regulated during state of traumatic injury.It may be of use to provide some strategies targeting the special nutrient requirements and transport capabilities of seriously traumatized patients.