Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7.
Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7.
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DOI：
10.1038/s41598-017-03146-5
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发表时间：
2017-06-06
影响因子：
4.6
通讯作者：
Tada M
中科院分区：
文献类型：
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作者：
Ueyama T;Tsuji S;Sugiyama T;Tada M
在早期药物发现和开发中，原代人肝细胞对于评价候选化合物的细胞毒性、药物代谢和药物-药物相互作用是必要的。然而，这些分析往往受到有限的资源和功能或遗传变异之间的地段。HepaRG人肝细胞癌细胞可以分化为具有与人肝细胞相似代谢活性的成熟肝细胞样细胞（HepLC）。我们先前建立了携带双报告基因的转基因HepaRG细胞，其在成肝细胞样细胞状态下在CYP 3A 7的转录调控下表达红色荧光蛋白（RFP），并在HepLC分化后在CYP 3A 4的转录调控下表达增强的绿色荧光蛋白（EGFP）。在这项研究中，我们成功地分离了一个亚克隆的转基因CYP 3A 4G/7 R HepaRG细胞与改善HepLC分化的潜力。在这些HepLC中通过CYP 3A 4的咪达唑仑代谢与野生型HepLC中相当。利福平（RIF）处理后，EGFP荧光强度显著增强。荧光分析和代谢分析之间有很强的相关性。EGFP阳性细胞的倍数变化与CYP 3A 4 mRNA水平和代谢产物中脯氨酸发光的倍数变化相当。RIF处理和细胞增殖增加RFP阳性细胞数量。因此，CYP 3A 4G/7 R HepLC提供了一种实时、基于多孔的系统来共同评价CYP 3A 4诱导和肝再生。
Primary human hepatocytes are necessary to evaluate cytotoxicity, drug metabolism, and drug–drug interactions for candidate compounds in early-phase drug discovery and development. However, these analyses are often hampered by limited resources and functional or genetic variation among lots. HepaRG human hepatocellular carcinoma cells can differentiate into mature hepatocyte-like cells (HepLCs) that possess similar metabolic activity to human hepatocytes. We previously established transgenic HepaRG cells carrying a dual reporter that express red fluorescent protein (RFP) under the transcriptional regulation of CYP3A7 in the hepatoblast-like cell state and enhanced green fluorescent protein (EGFP) under the transcriptional regulation of CYP3A4 following HepLC differentiation. In this study, we successfully isolated a subclone of transgenic CYP3A4G/7R HepaRG cells with an improved HepLC differentiation potency. Midazolam metabolism by CYP3A4 in these HepLCs was comparable to that in wild-type HepLCs. The EGFP fluorescence intensity was greatly induced by rifampicin (RIF) treatment. There was a strong correlation between fluorometric and metabolic analyses. The fold change in EGFP-positive cells was comparable to those in the CYP3A4 mRNA level and luminescence of proluciferin metabolites. RIF treatment and cell proliferation increased the RFP-positive cell number. Thus, CYP3A4G/7R HepLCs provide a real-time, multiwell-based system to co-evaluate CYP3A4 induction and hepatic regeneration.