Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism.
Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism.
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DOI：
10.1111/jcmm.13225
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发表时间：
2017-12
影响因子：
5.3
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Wang Y;Chang L;Zhai J;Wu Q;Wang D;Wang Y
原代肝细胞和干细胞来源的肝细胞样细胞（HLC）是生物人工肝（BAL）的主要来源。维持肝细胞功能和诱导HLCs功能成熟是BAL支持作用的关键。它仍然难以评估和改善肝细胞固有的解毒功能，包括氨清除。在这里，我们的目标是评估氨代谢和确定氨解毒增强剂通过开发一种成像策略。在肝癌细胞系HepG 2和永生化肝细胞系LO 2中，氨甲酰磷酸合成酶1（CPS 1）基因，氨消除尿素循环的第一种酶，通过CRISPR/Cas9系统用荧光蛋白标记。使用基于报告基因的筛选方法，从182种小分子的集合中选择细胞解毒增强剂。在两种CPS 1报告细胞系中，荧光强度与细胞CPS 1 mRNA表达、氨消除和分泌的尿素正相关，并以剂量依赖性方式反映氨解毒。令人惊讶的是，高水平的CPS 1报告基因克隆还保留了许多其他关键的肝细胞功能，例如白蛋白分泌和细胞色素450代谢功能。苯丁酸钠和白藜芦醇被鉴定为增强代谢相关基因表达和肝富集转录因子C/EBPα、HNF 4 α。总之，CPS 1报告基因系统提供了一个经济有效的平台，用于评估细胞代谢功能和高通量鉴定可改善肝系细胞解毒活性的化合物。
Both primary hepatocytes and stem cells‐derived hepatocyte‐like cells (HLCs) are major sources for bioartificial liver (BAL). Maintenance of hepatocellular functions and induction of functional maturity of HLCs are critical for BAL's support effect. It remains difficult to assess and improve detoxification functions inherent to hepatocytes, including ammonia clearance. Here, we aim to assess ammonia metabolism and identify ammonia detoxification enhancer by developing an imaging strategy. In hepatoma cell line HepG2, and immortalized hepatic cell line LO2, carbamoyl phosphate synthetase 1 (CPS1) gene, the first enzyme of ammonia‐eliminating urea cycle, was labelled with fluorescence protein via CRISPR/Cas9 system. With the reporter‐based screening approach, cellular detoxification enhancers were selected among a collection of 182 small molecules. In both CPS1 reporter cell lines, the fluorescence intensity is positively correlated with cellular CPS1 mRNA expression, ammonia elimination and secreted urea, and reflected ammonia detoxification in a dose‐dependent manner. Surprisingly, high‐level CPS1 reporter clones also reserved many other critical hepatocellular functions, for example albumin secretion and cytochrome 450 metabolic functions. Sodium phenylbutyrate and resveratrol were identified to enhance metabolism‐related gene expression and liver‐enriched transcription factors C/EBPα, HNF4α. In conclusion, the CPS1‐reporter system provides an economic and effective platform for assessment of cellular metabolic function and high‐throughput identification of chemical compounds that improve detoxification activities in hepatic lineage cells.
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影响因子：
13.5
作者：
Schofield, JP;Cox, TM;Wakamiya, M
通讯作者：
Wakamiya, M
DOI：
10.1152/ajpgi.00244.2014
发表时间：
2014-11-15
影响因子：
4.5
作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
4.8
作者：
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通讯作者：
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影响因子：
6
作者：
Liu, Hongyan;Dong, Huijia;Liu, Chen
通讯作者：
Liu, Chen