Cells for bioartificial liver devices: The human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia
Cells for bioartificial liver devices: The human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia
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DOI：
10.1002/bit.21599
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发表时间：
2008-02-15
影响因子：
3.8
通讯作者：
Hodgson, Humphrey J. F.
中科院分区：
文献类型：
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作者：
Mavri-Damelin, Demetra;Damelin, Leonard H.;Hodgson, Humphrey J. F.
肝外生物人工肝装置应提供完整的尿素循环来解毒氨。C3 A细胞系是肝癌衍生的HepG 2细胞系的亚克隆，目前用于这种情况下，因为它产生尿素，这被认为是通过功能性尿素循环的氨解毒的反映。然而，基于我们先前在不产生尿素的HepG 2细胞系中尿素循环功能紊乱的发现，我们假设C3 A细胞产生的尿素是通过尿素循环独立机制，即，由于尿素酶11活性，因此不会使氨解毒。使用N-15-氯化铵代谢标记和气相色谱-质谱法定量尿素。通过实时逆转录酶-PCR测定基因表达，通过蛋白质印迹法测定蛋白质表达，并通过放射性标记酶测定测定功能活性。精氨酸酶抑制研究使用N-ω-羟基-去甲-L-精氨酸。在C3 A条件培养基中检测到尿素;然而，15 N-氯化铵标记表明N-15-氨未掺入N-15-标记的尿素中。此外，基因表达的两个尿素循环基因，鸟氨酸转氨甲酰酶和辅酶1，完全不存在。相反，在C3 A细胞中，辅酶II mRNA和蛋白质以高水平表达，并被N-ω-羟基-去甲-L-精氨酸抑制，从而阻止尿素。生产，从而指示尿素循环独立途径。尿素循环在C3 A细胞中是无功能的，并且它们的尿素产生仅仅是由于。因此，其不能在生物人工肝系统中提供氨解毒。这强调了持续需要开发一种能够完全保留肝功能的成分。
Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase 11 activity, and therefore would not detoxify ammonia. Urea was quantified using N-15-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N-omega-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, 15N-ammonium chloride-labelling indicated that N-15-ammonia was not incorporated into N-15-labetled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase 1, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N-omega-hydroxy-nor-L-arginine, which prevented urea. production, thereby indicating a urea cycleindependent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due. to the presence of arginase 11, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function.