Recapitulation of metabolic defects in a model of propionic acidemia using patient-derived primary hepatocytes
Recapitulation of metabolic defects in a model of propionic acidemia using patient-derived primary hepatocytes
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DOI：
10.1016/j.ymgme.2015.12.008
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发表时间：
2016-03-01
影响因子：
3.8
通讯作者：
Dash, Ajit
中科院分区：
文献类型：
--
作者：
Chapman, Kimberly A.;Collado, Maria S.;Dash, Ajit
背景：丙酸血症（proonic acid mia， PA）是一种中间代谢紊乱，与丙酰辅酶a羧化酶（分别为PCCA和PCCB）酶的α或β亚基缺陷有关。我们之前描述了一种肝脏培养系统，该系统利用肝脏来源的血流动力学和运输参数来恢复和维持原代人肝细胞的生物学和代谢，利用生理相关的环境浓度。方法：在本研究中，从一名8岁PA患者的外植肝脏中分离出原代肝细胞，在肝脏系统中培养10天，并评估分化极化形态的保留情况。相对于健康供体对照，在基因和蛋白水平上评估PCCA和PCCB的表达。在存在和不存在氨基酸补充剂的情况下测量氨和尿素水平，以评估支链氨基酸代谢在该疾病中的代谢后果。结果：PA患者的原代肝细胞在系统中培养10天后保持分化极化形态（外周肌动蛋白染色）。我们注意到PCCA和PCCB在mRNA和蛋白质水平上相对于正常健康对照的水平较低。在培养基中补充支链氨基酸异亮氨酸（5mm）和缬氨酸（5mm），导致PA患者肝细胞系统中氨增加和尿素减少，但在健康肝细胞或患者来源的成纤维细胞中未见这种反应。结论：我们首次成功培养了分化状态的PA患者源性原代肝细胞，在基因和蛋白水平上稳定地保留了PCCA和PCCB酶缺陷。该系统对支链氨基酸负荷增加的表型反应，在成纤维细胞中是不可能的，强调了该系统在更好地理解PA的分子病理生理和检查潜在治疗剂在最相关组织中的有效性方面的效用。(c) 2015 Elsevier Inc.版权所有。
Background: Propionic acidemia (PA) is a disorder of intermediary metabolism with defects in the alpha or beta subunits of propionyl CoA carboxylase (PCCA and PCCB respectively) enzyme. We previously described a liver culture system that uses liver-derived hemodynamic blood flow and transport parameters to restore and maintain primary human hepatocyte biology and metabolism utilizing physiologically relevant milieu concentrations.Methods: In this study, primary hepatocytes isolated from the explanted liver of an 8-year-old PA patient were cultured in the liver system for 10 days and evaluated for retention of differentiated polarized morphology. The expression of PCCA and PCCB was assessed at a gene and protein level relative to healthy donor controls. Ammonia and urea levels were measured in the presence and absence of amino acid supplements to assess the metabolic consequences of branched-chain amino acid metabolism in this disease.Results: Primary hepatocytes from the PA patient maintained a differentiated polarized morphology (peripheral actin staining) over 10 days of culture in the system. We noted lower levels of PCCA and PCCB relative to normal healthy controls at the mRNA and protein level. Supplementation of branched-chain amino acids, isoleucine (5 mM) and valine (5 mM) in the medium, resulted in increased ammonia and decreased urea in the PA patient hepatocyte system, but no such response was seen in healthy hepatocytes or patient derived fibroblasts.Conclusions: We demonstrate for the first time the successful culture of PA patient-derived primary hepatocytes in a differentiated state, that stably retain the PCCA and PCCB enzyme defects at a gene and protein level. Phenotypic response of the system to an increased load of branched-chain amino acids, not possible with fibroblasts, underscores the utility of this system in the better understanding of the molecular pathophysiology of PA and examining the effectiveness of potential therapeutic agents in the most relevant tissue. (c) 2015 Elsevier Inc. All rights reserved.