Substrate stiffness regulates primary hepatocyte functions
Substrate stiffness regulates primary hepatocyte functions
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DOI：
10.1039/c5ra15208a
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发表时间：
2015-01-01
期刊：
影响因子：
3.9
通讯作者：
Kidambi, Srivatsan
中科院分区：
文献类型：
--
作者：
Natarajan, Vaishaali;Berglund, Eric J.;Kidambi, Srivatsan
肝纤维化的发生是由于病毒感染、代谢紊乱和酒精滥用造成的慢性损伤。纤维化肝脏微环境（LME）的特征是细胞外基质蛋白的过度沉积和异常周转，这导致组织硬度增加。肝硬度在健康和疾病状态下都是调节肝脏反应的重要线索;然而，不同硬度对肝细胞的影响还不清楚。迫切需要设计模拟对应于疾病进展的各个阶段的肝硬度的体外模型，以阐明个体细胞反应的作用。在这里，我们采用聚二甲基硅氧烷（PDMS）为基础的基板与可调的机械性能，研究基板刚度对原代大鼠肝细胞的行为的影响。为了重建生理相关的硬度，我们设计了软基质（2 kPa）来代表健康的肝脏，硬基质（55 kPa）来代表患病的肝脏。组织培养板表面（TCPS）作为对照基质。我们观察到，与硬基质和TCPS相比，在软基质上培养的肝细胞在更长的时间内显示出更分化和功能性的表型。我们证明了软基质上的肝细胞具有较高的尿素和白蛋白合成。细胞色素P450（CYP）活性，肝细胞的另一个关键标志物，显示出对基质硬度的强烈依赖性，其中与TCPS相比，软基质上的肝细胞在培养第7天保留了2.7倍的CYP活性。我们进一步观察到刚度的增加诱导了关键药物转运蛋白基因（NTCP，UGT 1A 1和GSTM-2）的下调。此外，我们观察到，上皮细胞表型更好地保持软基板上的肝细胞核因子4 α，细胞角蛋白18和连接蛋白32的较高表达所示。这些结果表明，基质硬度在调节肝细胞行为中起着重要作用。我们的基于PDMS的肝脏模型可用于研究介导肝细胞-LME通讯的信号通路，以了解肝脏疾病的进展。
Liver fibrosis occurs as a consequence of chronic injuries from viral infections, metabolic disorders, and alcohol abuse. Fibrotic liver microenvironment (LME) is characterized by excessive deposition and aberrant turnover of extracellular matrix proteins, which leads to increased tissue stiffness. Liver stiffness acts as a vital cue in the regulation of hepatic responses in both healthy and diseased states; however, the effect of varying stiffness on liver cells is not well understood. There is a critical need to engineer in vitro models that mimic the liver stiffness corresponding to various stages of disease progression in order to elucidate the role of individual cellular responses. Here we employed polydimethyl siloxane (PDMS) based substrates with tunable mechanical properties to investigate the effect of substrate stiffness on the behavior of primary rat hepatocytes. To recreate physiologically relevant stiffness, we designed soft substrates (2 kPa) to represent the healthy liver and stiff substrates (55 kPa) to represent the diseased liver. Tissue culture plate surface (TCPS) served as the control substrate. We observed that hepatocytes cultured on soft substrates displayed a more differentiated and functional phenotype for a longer duration as compared to stiff substrates and TCPS. We demonstrated that hepatocytes on soft substrates exhibited higher urea and albumin synthesis. Cytochrome P450 (CYP) activity, another critical marker of hepatocytes, displayed a strong dependence on substrate stiffness, wherein hepatocytes on soft substrates retained 2.7 fold higher CYP activity on day 7 in culture, as compared to TCPS. We further observed that an increase in stiffness induced downregulation of key drug transporter genes (NTCP, UGT1A1, and GSTM-2). In addition, we observed that the epithelial cell phenotype was better maintained on soft substrates as indicated by higher expression of hepatocyte nuclear factor 4 alpha, cytokeratin 18, and connexin 32. These results indicate that the substrate stiffness plays a significant role in modulating hepatocyte behavior. Our PDMS based liver model can be utilized to investigate the signaling pathways mediating the hepatocyte-LME communication to understand the progression of liver diseases.