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首页> 外文期刊>Cellular Physiology and Biochemistry >Targeting Nuclear Receptors with Lentivirus-Delivered Small RNAs in Primary Human Hepatocytes
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Targeting Nuclear Receptors with Lentivirus-Delivered Small RNAs in Primary Human Hepatocytes

机译：在原代人肝细胞中以慢病毒传递的小RNA靶向核受体。

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biBackground /i/bRNA interference (RNAi) has tremendous potential for investigating gene function and for developing new therapies. Primary human hepatocytes (PHH) are the “gold standard” for studying the regulation of hepatic metabolism iin vitro/i. However, application of RNAi in PHH has some technical hurdles. The objective of this study was to develop effective and robust protocol for transduction of PHH with lentiviral vectors. biMethods /i/bWe used lentiviral vectors to transduce PHH for introduction of short hairpin RNAs (shRNAs) targeting constitutive androstane receptor (CAR), peroxisome proliferator activated receptor alpha (PPARα), and microRNA, miR-143. Infection efficiency was quantitatively analyzed by flow cytometry and microscopy. Target gene expression was assessed using quantitative real-time (qRT-PCR) method. biResults /i/bLentiviral vector transduction resulted in ≥95% of infected cells at low multiplicity of infection (MOI) of 3, which did not impair cellular viability. We demonstrated the feasibility of this technique in studies on targeting nuclear receptors, PPARα and CAR, with shRNAs as well as in lentivirus-mediated overexpression and knock-down of miRNA-143 experiments. biConclusions /i/bWe developed an efficient and robust protocol with standardized procedures for virus production, method of titer determination, and infection procedure for RNAi in primary human hepatocytes based on delivery of shRNAs, microRNAs or anti-microRNAs in different laboratory settings. This approach should be useful to study not only the regulation via nuclear receptors but also other biological, pharmacological, and toxicological aspects of drug metabolism.
机译：背景 RNA干扰（RNAi）在研究基因功能和开发新疗法方面具有巨大潜力。人类原代肝细胞（PHH）是体外研究肝脏代谢调控的“金标准”。然而，RNAi在PHH中的应用存在一些技术障碍。这项研究的目的是开发有效和鲁棒的协议，以慢病毒载体转导PHH。 方法 我们使用慢病毒载体转导PHH，以导入针对组成型雄激素受体（CAR），过氧化物酶体增殖物激活受体α（PPARα）和microRNA的短发夹RNA（shRNA） ，miR-143。通过流式细胞术和显微镜术定量分析感染效率。使用定量实时（qRT-PCR）方法评估靶基因的表达。 结果 慢病毒载体转导在低感染复数（MOI）为3时导致≥95％的被感染细胞，这并不损害细胞活力。我们证明了该技术在针对靶向shRNA的核受体，PPARα和CAR以及慢病毒介导的miRNA-143实验过表达和敲低研究中的可行性。 结论 我们基于shRNA，microRNA的传递，开发了一种高效且强大的协议，其中包括用于生产病毒，标准滴度确定方法以及人类原代肝细胞中RNAi感染程序的标准化程序或在不同实验室环境中使用抗microRNA。这种方法不仅对研究通过核受体的调节，而且对药物代谢的其他生物学，药理学和毒理学方面都应是有用的。

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