BioMediTech, Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland.
Zora Biosciences, 02150 Espoo, Finland.
Int J Mol Sci. 2019 Jun 14;20(12):2910. doi: 10.3390/ijms20122910.
Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated.
PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR.
The typical morphology of PHHs was lost from day 3 onward. Additionally, and genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, , , , , and were upregulated but was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation.
Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs' loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.
原代人肝细胞（PHH）在二维（2D）培养中经历去分化，这特别阻碍了它们在长期体外研究中的应用。脂质作为一大类生物分子，在细胞能量储存、结构和信号传递中起着至关重要的作用。在这里，我们首次绘制了去分化 PHH 中脂质谱的变化，并研究了脂质在 PHH 表型丧失中的可能作用。同时，研究了与去分化 PHH 中脂质和脂肪酸（FA）变化相关的差异表达 miRNA。
将 PHH 在单层中培养，并从形态学、遗传学和生化方面监测其表型，为期五天。通过质谱和安捷伦微阵列分别分析 PHH 的脂质和 miRNA 谱。此外，通过 qPCR 研究了 24 个涉及脂质和 FA 代谢的关键基因。
PHH 的典型形态从第 3 天开始丧失。此外，培养的 PHH 中 和 基因下调。脂质组学显示，在 PHH 的去分化过程中，饱和脂肪酸（SFA）和单不饱和脂肪酸（MUFA）含量增加的脂质明显增加，而多不饱和脂肪酸（PUFA）含量减少的脂质明显增加。与此一致的是， 、 、 、 、 和 在去分化 PHH 中上调，但 下调。此外，还鉴定出差异表达的 miRNA，不断上调的 miR-27a 和 miR-21 以及下调的 miR-30 可能在 PHH 脂质的去分化过程中调节其合成、积累和分泌。
我们的研究结果表明，在 PHH 长时间培养过程中，其分子脂质种类谱、脂质代谢酶表达以及 miRNA 谱发生了重大变化，这些变化共同作用可能在 PHH 表型丧失中发挥重要作用。这些发现促进了对去分化过程的理解，并有助于开发更好地满足 PHH 需求并支持其原始表型的最佳培养条件。
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