Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.
Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.
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DOI：
10.1007/s00204-016-1694-y
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发表时间：
2017-01
影响因子：
6.1
通讯作者：
Kevin Park B
中科院分区：
文献类型：
--
作者：
Heslop JA;Rowe C;Walsh J;Sison-Young R;Jenkins R;Kamalian L;Kia R;Hay D;Jones RP;Malik HZ;Fenwick S;Chadwick AE;Mills J;Kitteringham NR;Goldring CE;Kevin Park B
从人体组织中分离出原代人肝细胞后的应用被公认为会受到去分化过程的影响。这种现象降低了许多独特的肝细胞功能，限制了它们在药物处置和毒性评估中的使用。去分化的病因还没有很好的定义，进一步了解这一过程将有助于开发新的策略来维持培养中的肝细胞表型，或者改进干细胞产生的肝细胞成熟的方案。因此，我们首次进行了人类原代肝细胞分化的蛋白质组学比较。细胞培养0、24、72和168h作为单层细胞，以允许无限制的肝细胞去分化，从而通过通路分析揭示这一过程中的致病信号通路和因素。共鉴定出3430个蛋白质，误检率为1%，其中1117个蛋白质在每个时间点都进行了量化。与新鲜分离的细胞相比，在24小时(40个蛋白质)、72小时(118个蛋白质)和168小时(272个蛋白质)显著差异表达的蛋白质数目增加(P<0.05)。特别是，细胞色素P450和线粒体蛋白发生了重大变化，功能研究证实了这一点，并通过通路分析进行了研究。我们报告了在体外导致肝脏表型丧失的关键因素和途径，特别是那些驱动线粒体和代谢蛋白质组大规模和选择性重塑的因素和途径。总之，这些发现扩大了目前对去分化的理解，应该有助于进一步开发简单和复杂的肝脏培养系统。本文的在线版本(doi：10.1007/s00204-0161694-y)包含补充材料，可供授权用户使用。
The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems. The online version of this article (doi:10.1007/s00204-016-1694-y) contains supplementary material, which is available to authorized users.