肝细胞体外培养去分化过程中转录因子表达谱分析

Analysis of transcriptional factors expression profile during dedifferentiation of liver cell cultured in vitro

摘要:

原代肝实质细胞被广泛应用到药物代谢和毒性评估中，但其体外培养呈现去分化状态，表现为逐渐失去正常形态和代谢解毒功能，到目前为止对去分化的分子机制并不清楚。在肝实质细胞体外去分化过程中转录因子 (Transcription factor，TF) 起重要作用，而且非实质细胞可以在体外维持肝实质细胞功能。然而目前的技术手段不能有效地鉴定和定量分析大量TFs。本文建立了单层肝实质细胞单独培养以及肝实质细胞与非实质细胞共培养系统，细胞培养到24、48、72 h。利用TF富集技术 (Transcription factor response elements on tip，TOT) 和质谱技术研究在肝实质细胞体外培养过程中TFs变化。本研究3次重复实验共鉴定到219个TFs，分析发现肝实质细胞培养过程中与细胞增殖、死亡、免疫等通路相关的TFs表达增高，而与代谢通路相关的TFs表达减弱。我们建立的肝细胞培养-TFs鉴定系统对揭示肝实质细胞去分化的分子机制以及肝实质与非实质细胞间相互作用具有重要意义。

Abstract:

Primary hepatocytes are widely used in drug metabolism and toxicity assessment. As the culture of primary hepatocytes in vitro is a process of dedifferentiation, hepatocytes lose normal metabolic detoxification function gradually. The mechanism of hepatocyte dedifferentiation has been not clear so far. TFs play an important role in the dedifferentiation and non-parenchymal cells can maintain the function of hepatocytes in vitro. However, the current methods cannot be used in effective identification and quantitative analysis of a large number of TFs. In this paper, the mo-culture system (only primary hepatocytes) and co-culture system (primary hepatocytes and non-parenchymal cells) were established. The cells were cultured for 24 h, 48 h, 72 h as monolayer. The changes of TFs during the culture were obtained by TOT (Transcription factor response elements on tip) transcription factor enrichment method and mass spectrometry. A total of 219 TFs were identified in three individual replicates. The result revealed that up-regulated TFs were enriched in cell proliferation, death and immune response pathways, and down-regulated TFs were involved in metabolism pathway. The establishment of such culture-TFs identification system is of great significance to reveal the mechanism of primary hepatocyte dedifferentiation and crosstalk between hepatocytes and non-parenchymal cells.