优化分析<em>在体内</em>和<em>体外</em>脂肪肝
Summary
Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.
Abstract
Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models.
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.
Introduction
肥胖是发达国家和发展中国家一个新兴的健康问题。据报道是经常与非酒精性脂肪肝病（NAFLD）相关联的共存条件之一，具有患病30％和100％的NAFLD患者1之间的范围内。由于脂肪肝和肥胖症之间的强相关性，膳食诱导的肥胖（DIO）小鼠模型被广泛用于研究与NAFLD 2，3，4，5，6的发展相关联的复杂的分子机制。肝脂肪变性是NAFLD的最早阶段，它可以前进到非酒精性脂肪性肝炎（NASH），肝硬化，最终，肝癌7。因此，这种方法的总体目标是产生的肝脂肪变性条件动物和细胞模型和PR奥维德的高效，准确的测量血脂详细的协议。这些模型和测量也可用于其他代谢疾病，如胰岛素抵抗和2型糖尿病的调查是有用的。
由于肥胖被确定成为NAFLD，高脂肪的主要危险因素之一，高糖饮食（HFHS）模仿西式高脂饮食是用于诱导的肥胖小鼠。接着，肝脂肪变性的程度可以用不同的方法来评估。首先，体重和核磁共振（NMR）身体成分的分析表明喂养时间内脂质积累。的脂肪量和瘦体重可以以非侵入性的和实时的方式进行量化。
此外，磁共振成像（MRI）是用来同时显示全身和脂肪肝的分布。在MRI分析的灰度级信号可以被转换成清晰的伪彩色图象，和强度在灰度和彩色是半定量的。该技术提供了独特的优势，为在活的动物脂肪积累的测量。第二，肝脏的组织学分析是最常用的方法来确定肝脂肪变性。苏木精和曙红（H＆E）染色的组织学提供的信息，如肝细胞形态和巨噬细胞浸润，而油红O染色显示肝细胞中脂质小滴的大小和位置。第三，用氯仿/甲醇萃取脂质含量分析是肝脂质的精确和定量的测量。总甘油三酯和胆固醇水平可与生化方法来测量。重要的是，脂质提取分析和油红O染色，也可在基因操作或药学治疗肝细胞使用。
本方法的优点是多种优化方法及其利用，以产生肝脂肪变性模型和全面表征表型在体内和体外 。该DIO小鼠模型可以概括人类脂肪肝疾病的病理和代谢表型。在人类其他代谢参数可以在这个模型以及8被复制。响应于高糖加胰岛素的脂肪肝的肝细胞模型的生成是有效的，有用的，并且克服了昂贵和耗时鼠标工作的限制。两者合计，这些方法是足够和必不可少的肝脏脂质功能障碍和胰岛素抵抗养分超载在研究。
Protocol
Representative Results
Discussion
NAFLD是一系列与代谢综合征，肥胖，胰岛素抵抗或II型糖尿病（T2DM）11相关联的进行性肝脏疾病。 NAFLD的特点是脂肪变性，脂质在肝细胞中的积累。这里，提出来表征表型和使用的DIO小鼠和小鼠原代肝细胞脂肪肝的参数的方法的光谱。此过程可帮助阐明NAFLD和其他相关的代谢性疾病的分子机制。
在体内和体外实验相结合，这些方法提供了全面的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.
Materials
| O.C.T compound | SAKURA | 4583 | |
| Oil Red O | Sigma | O0625-25G | |
| Infinity Triglycerides kit | Fisher Scientific | TR22421 | |
| Infinity Cholesterol kit | Fisher Scientific | TR13421 | |
| Collagen type I, Rat tail | Millipore | 08-115 | |
| DMEM (low glucose) | Invitrogen | 11885-092 | |
| Penicillin / Streptomycin | Invitrogen | 15140-122 | |
| FBS | Invitrogen | 10099141 | |
| PBS | cellgro | R21-040-CVR | |
| HBSS | cellgro | 20-021-CV | |
| Insulin | TOCRIS Bioscience | 3435 | dissolve in PBS, 1mM for stock |
| Glucose | Sigma | G8270-100G | |
| Microscope | Olympus | BX53 | |
| Peristaltic pump | Longerpump | BT100-2J | |
| 10cm cell culture dish | Corning | 420167 | |
| 6-well-plate | Corning | 3516 | |
| BCA assay | Beyotime | P0010 | |
| Nuclear Magnetic Resonance | Niumag technology | MiniQMR23-060H-I | |
| High fat high surcose diet(HFHS) | Research Diets | D12327 |
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