Development of an in vitro cell culture model of hepatic steatosis using hepatocyte-derived reporter cells.
Development of an in vitro cell culture model of hepatic steatosis using hepatocyte-derived reporter cells.
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DOI：
10.1002/bit.22191
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发表时间：
2009-04-01
影响因子：
3.8
通讯作者：
Yarmush, Martin L.
中科院分区：
文献类型：
--
作者：
Janorkar, Amol V.;King, Kevin R.;Megeed, Zaki;Yarmush, Martin L.
脂肪肝是一个日益重要的临床问题，因为它与日益普遍的肥胖和糖尿病有关。虽然脂肪变性代表肝内脂质过量的可逆状态，但它也与对氧化和细胞因子应激的易感性增加以及进展为以脂肪性肝炎、肝硬化和恶性肿瘤为特征的不可逆肝损伤相关。目前，这种动态疾病进展的分子机制仍然知之甚少，特别是在转录调控水平。我们最近构建了一个稳定的单克隆绿色荧光蛋白（GFP）报告细胞，使转录调控在活细胞中进行动态研究库。在这里，我们调整报告细胞，以创建一个模型，脂肪变性，将允许调查与脂肪变性的发展和随后的“二次打击”应力的反应相关的转录动力学。报告模型概括了人类疾病的许多细胞特征，包括脂肪酸摄取、细胞内甘油三酯积累、活性氧积累增加、线粒体膜电位降低、对凋亡细胞因子应激的易感性增加和增殖降低。最后，为了证明报告细胞在研究转录调节方面的实用性，我们比较了核因子κB（NFκB）、热休克反应元件（HSE）和糖皮质激素反应元件（GRE）在瘦肉和脂肪条件下响应其经典诱导剂的转录动力学，发现细胞内脂质积累与NFκB和HSE的剂量依赖性损伤有关，但与GRE激活无关。因此，脂肪变性报告细胞是研究转录反应的有效模型，并有可能为脂肪肝疾病的进展提供重要的见解。
Fatty liver disease is a problem of growing clinical importance due to its association with the increasingly prevalent conditions of obesity and diabetes. While steatosis represents a reversible state of excess intrahepatic lipid, it is also associated with increased susceptibility to oxidative and cytokine stresses and progression to irreversible hepatic injury characterized by steatohepatitis, cirrhosis, and malignancy. Currently, the molecular mechanisms underlying progression of this dynamic disease remain poorly understood, particularly at the level of transcriptional regulation. We recently constructed a library of stable monoclonal green fluorescent protein (GFP) reporter cells that enable transcriptional regulation to be studied dynamically in living cells. Here, we adapt the reporter cells to create a model of steatosis that will allow investigation of transcriptional dynamics associated with the development of steatosis and the response to subsequent “second hit” stresses. The reporter model recapitulates many cellular features of the human disease, including fatty acid uptake, intracellular triglyceride accumulation, increased reactive oxygen species accumulation, decreased mitochondrial membrane potential, increased susceptibility to apoptotic cytokine stresses, and decreased proliferation. Finally, to demonstrate the utility of the reporter cells for studying transcriptional regulation, we compared the transcriptional dynamics of nuclear factor κB (NFκB), heat shock response element (HSE), and glucocorticoid response element (GRE) in response to their classical inducers under lean and fatty conditions and found that intracellular lipid accumulation was associated with dose-dependent impairment of NFκB and HSE but not GRE activation. Thus, steatotic reporter cells represent an efficient model for studying transcriptional responses and have the potential to provide important insights into the progression of fatty liver disease.
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影响因子：
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Bianchi, A;Bécuwe, P;Dauça, M
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