Name: in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease
Uploaded: 2024-07-19T14:10:00.000+00:00
Duration: 7 min 3 s
Description: The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

本研究获内蒙古自治区科技厅科技计划2018年支持项目"Koumiss对蒙古医药地方病疗效关键问题研究"资助。

注:有关本协议中使用的所有材料、仪器和试剂的详细信息，请参阅 材料表 。
1. 细胞培养
<ol><li>在含有 Dulbecco 改良 Eagle 培养基 （DMEM）（含有 10% 胎牛血清 [FBS]、100 单位/mL 青霉素和 100 μg/mL 链霉素）的培养瓶中培养 HepG2 细胞。将培养瓶保持在 37 °C 的 5% CO2 培养箱中。</li>
</ol>2. 通过细胞计数试剂盒 8 测量油酸对细胞活力的影响
<ol><li>将特定体积的 OA 溶解在二甲基亚砜 （DMSO） 中，以达到 200 mM 的浓度。将溶液储存在 -20 °C 以备将来使用。</li>
	<li>将 HepG2 细胞以每孔 6 × 103 个细胞的细胞密度接种在 96 孔板中。向每个孔中加入 100 μL DMEM。将细胞在 37 °C 下在 5% CO2 培养箱中孵育和培养 24 小时。将 HepG2 细胞分为两组:<ol><li>对照组:添加细胞培养基。</li>
		<li>OA 组:向细胞培养基中加入 OA，使最终浓度达到 0.125 mM、0.25 mM、0.5 mM 和 1 mM。</li>
	</ol></li>
	<li>初始孵育 24 小时后，弃去每个孔中的上清液，并根据指定的分组加入 OA，每孔添加 100 μL。对于对照组，向每个孔中加入 100 μL 细胞培养基。继续将细胞再孵育 24 小时。 注意:确保每组有六个重复孔。为防止蒸发，向 96 孔板的孔外环中加入 100 μL 磷酸盐缓冲盐水 （PBS）。</li>
	<li>孵育 24 小时后，向每个孔中加入 10 μL 细胞计数试剂盒-8 （CCK-8），轻轻混匀，并在黑暗中孵育 2 小时。从培养箱中取出 96 孔板，将其放入酶标仪中，并测量 450 nm 处的吸光度值 （A450）。根据 OD 计数 GI50 值。</li>
</ol>3. 油红 O 染色观察细胞内脂滴形成
<ol><li>将 HepG2 细胞以每孔 5 × 105 个细胞 的密度接种在 6 孔细胞培养板中，并在恒温培养箱中培养板 24 小时。请参阅 图 1 了解所描述步骤的直观表示。</li>
	<li>细胞培养 24 小时后，向每个孔中加入 2 mL 含有 OA 的细胞培养基，最终浓度达到 0.125 mM、0.25 mM、0.5 mM 和 1 mM。再过 24 小时后，从每个孔中取出细胞培养基，并用 PBS 洗涤两次。向每个孔中加入 1 mL ORO 固定剂并孵育 30 分钟。</li>
	<li>通过将染色液 A 与染色液 B 以 3:2 的比例混合来制备 ORO 染色液。让混合物在室温下静置 10 分钟，然后通过 0.45 μm 过滤器过滤一次。将过滤后的溶液储存在避光的离心管中，直至使用。</li>
	<li>丢弃固定剂并用蒸馏水洗涤两次。向每个孔中加入 1 mL 60% 异丙醇并孵育 30 秒。弃去 60% 的异丙醇溶液，并在孵育 20 分钟之前向每个孔中加入 1 mL 新鲜制备的 ORO 染色溶液。弃去 ORO 染色溶液，向每个孔中加入 1 mL 60% 异丙醇，并孵育 30 秒。用水洗涤 5 次以去除多余的染料。</li>
	<li>用蒸馏水覆盖细胞并在显微镜下观察。收集图像后，丢弃板中的液体并让其干燥。然后，向每个孔中加入 2 mL 异丙醇，并在轨道摇床上摇动板 10 分钟。将液体转移到新的 96 孔板中，每组 16 孔，每孔添加 100 μL。通过使用酶标仪在 510 nm 处测量每个孔的光密度 （OD） 来计算脂质含量 （A510）。</li>
</ol>4. 不同浓度油酸对 HepG2 细胞上清液中总甘油三酯的影响
<ol><li>在室温下平衡试剂盒 20 分钟，然后准备实验所需的板。</li>
	<li>收集细胞上清液并以 1,570 × g 离心 10 分钟。设置标准孔和测试样品孔。向标准孔中加入 50 μL 标准（[S0 → S5] 浓度，然后加入:0、0.5、1、2、4、8 mmol/L）。除了空白孔和标准孔外，向样品孔中加入 10 μL 不同的样品，然后向每个孔中加入 40 μL 样品稀释剂。向每个孔中加入 100 μL 检测抗体辣根过氧化物酶，用板膜密封，并在 37 °C 下在恒温箱中孵育 1 小时。</li>
	<li>弃去上清液，在无尘纸上吸干，并用 1x 洗涤液洗涤每个孔。在室温下静置 1 分钟。重复洗涤过程 5 次。</li>
	<li>向每个孔中加入 50 μL 底物 A 和 50 μL 底物 B。轻轻混匀并在 37 °C 下孵育 15 分钟。 向每个孔中加入 50 μL 终止溶液，并在 15 分钟内测量每个孔在 450 nm （A450） 处的 OD 值。</li>
	<li>沿 x 轴绘制标准品浓度，沿 y 轴绘制相应的吸光度 （OD） 值以执行线性回归，并推导出曲线方程以计算每个样品的浓度值。</li>
</ol>5. 统计分析
<ol><li>确定定量数据的显著差异。</li>
	<li>计算标准差 （SD） ±平均值并以图形方式表示数据。认为 P < 0.05 具有统计显著性。</li>
</ol>

用于代谢疾病分析的油酸诱导的 HepG2 细胞 

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oleic/palmitic+acid+imbalance+in+exosomes+isolated+from+NAFLD+patients+induces+necroptosis+of+liver+cells+via+the+elongase-6/RIP-1+pathway.">The oleic/palmitic acid imbalance in exosomes isolated from NAFLD patients induces necroptosis of liver cells via the elongase-6/RIP-1 pathway.</a> Cell Death Dis. 14 (9), 635 (2023).</li><li>Chavez-Tapia, N. C., Rosso, N., Tiribelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+intracellular+lipid+accumulation+in+a+new+model+of+non-alcoholic+fatty+liver+disease.">Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease.</a> BMC Gastroenterol. 12, 20 (2012).</li><li>Ricchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+oleic+and+palmitic+acid+on+lipid+accumulation+and+apoptosis+in+cultured+hepatocytes.">Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes.</a> J Gastroenterol Hepatol. 24 (5), 830-840 (2009).</li></ol>

作者声明没有利益冲突。

MASLD 是一种临床病理综合征，其特征是由于酒精和其他已确定的肝脏损伤因子以外的因素导致肝细胞中细胞内脂肪沉积过多18。MASLD 与获得性代谢应激性肝损伤有着错综复杂的联系，特别是与胰岛素抵抗和遗传易感性有关。为了有效地研究和筛选 MASLD 药物，选择合适的实验模型至关重要。建立细胞模型在 MASLD 研究中尤为重要，有助于更深入地了解病理机制和评估新药作用。
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代谢功能障碍相关脂肪性肝病 （MASLD） 包括一系列病症，包括单纯性脂肪变性、非酒精性脂肪性肝炎 （NASH）、肝硬化和肝细胞癌 1,2,3,4,5,6，所有这些都归因于酒精消耗以外的因素 7 .MASLD 是由代谢性肝损伤引起的最普遍的肝脏疾病，影响全球近四分之一的人口 8,9,10,11,12。虽然 MASLD 的确切发病机制尚未阐明，但各种理论试图解释其发展。一个流行的观点表明，从经典的 "two-hit" 理论转向 "multiple-hit" 模型1。这些假设的核心是胰岛素抵抗的作用，这被认为是 MASLD 发病机制的关键13。研究表明，肝细胞中的胰岛素抵抗会导致游离脂肪酸水平升高，随后形成储存在肝脏内的甘油三酯14,15。
研究人员已经使用 体内 和 体外 模型来模拟 MASLD 中的脂肪沉积;然而，完全复制其病理机制仍然具有挑战性。尽管存在这种限制，但这些模型有助于研究 MASLD 的潜在治疗靶点。然而，开发稳定的 MASLD 模型至关重要。虽然动物模型有效，但它们既耗时又昂贵，因此凸显了人们对 体外 细胞模型的兴趣日益浓厚。这些模型通常使用单一或多种游离脂肪酸，如油酸 （OA） 和棕榈酸）来重建饮食诱导的 MASLD。其中，人肝母细胞瘤细胞系 HepG2 常用于建立 MASLD 的 体外 细胞模型。
OA 诱导刺激 HepG2 细胞复制脂肪沉积，类似于 MASLD，这是一种具有悠久历史的方法。本研究的目的是证明用 0.25 mM OA 处理的 HepG2 细胞的活力、油红 O （ORO） 染色、脂质含量和甘油三酯 （TG） 水平。该实验的目的是为 MAFLD 建模研究的发展提供进一步的证据。

<table><tbody><tr><td>0.22 &amp;micro;m filter</td><td>Millex</td><td></td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x) Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>0.45 &amp;micro;m filter</td><td>Millex</td><td></td><td></td></tr><tr><td>2 mL Crygenic Vials</td><td>CORNING</td><td>430659</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Cell Culture Flask</td><td>CORNING</td><td>430639</td><td></td></tr><tr><td>6-well cell culture plate</td><td>CORNING</td><td>3516</td><td></td></tr><tr><td>96-well cell culture plate</td><td>CORNING</td><td>3599</td><td></td></tr><tr><td>Blood Count Plate</td><td>Shanghai Jing Jing Biochemical Reagent &amp;amp; Instrument Co.</td><td>02270113</td><td></td></tr><tr><td>Cell Counting Kit-8 assays</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</td><td>CA1210-1000T</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>NUAIRE</td><td>NU-5710E</td><td></td></tr><tr><td>&amp;nbsp;DMSO Dimethyl sulfoxide&amp;nbsp;</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</td><td>D8371</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco</td><td>8122691</td><td></td></tr><tr><td>Enzyme Labeling Equipment</td><td>Tecan</td><td>Spark</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified</td><td>Gibco</td><td>10099141</td><td></td></tr><tr><td>HepG2 cells line</td><td>Beijing North China Chuanglian Biotechnology Research Institute (BNCC)</td><td>221031</td><td></td></tr><tr><td>Human Triglyceride (TG) ELISA instruction</td><td>Nanjing Jiacheng Bioengineering Institute</td><td>20170301</td><td></td></tr><tr><td>Inverted Microscope for Cell Culture</td><td>Leica</td><td>DMi1&amp;nbsp;</td><td></td></tr><tr><td>Isopropanol</td><td>Tianjin Zhiyuan Chemical Reagent Co.</td><td>2021030141</td><td></td></tr><tr><td>Oil Red Stain Kit, For Cultured Cells</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</td><td>G1262</td><td></td></tr><tr><td>Oleic acid&amp;nbsp;</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A502071</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>SPSS 24.0</td><td></td><td>Statistics software</td><td></td></tr></tbody></table>

in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease

由于经济和生活方式的变化，代谢功能障碍相关脂肪性肝病 （MASLD） 的患病率激增，导致重大的健康挑战。以前的报告研究了 MASLD 的动物和细胞模型的建立，突出了它们之间的差异。在这项研究中，通过在 MASLD 中诱导脂肪堆积来创建一个细胞模型。用不同浓度 （0.125 mM、0.25 mM、0.5 mM、1 mM） 的不饱和脂肪酸油酸刺激 HepG2 细胞以模拟 MASLD。使用细胞计数试剂盒 8 测定、油红 O 染色和脂质含量分析评估模型的疗效。本研究旨在为 MASLD 细胞创建一个易于操作的细胞模型。细胞计数试剂盒 8 测定结果显示，HepG2 细胞的存活率取决于油酸的浓度，GI50 为 1.875 mM。0.5 mM 和 1 mM 组的细胞活力显著低于对照组 （P < 0.05）。此外，油红 O 染色和脂质含量分析检查了不同油酸浓度 （0.125 mM、0.25 mM、0.5 mM、1 mM） 在 HepG2 细胞上的脂肪沉积。0.25 mM 、 0.5 mM 和 1 mM 组的脂质含量显著高于对照组 （P < 0.05）。此外，OA 组甘油三酯水平显著高于对照组 （P < 0.05）。

油酸对细胞活力的影响 HepG2 细胞暴露于不同浓度的 OA （0 mM、0.125 mM、0.25 mM、0.5 mM、1 mM），导致细胞存活率在 0.125 mM、0.25 mM、0.5 mM 和 1 mM 时与 0 mM 相比降低。与 0 mM 相比，在 0.5 mM （P < 0.05） 和 1 mM （P < 0.05） 时观察到统计学意义。由 CCK-8 试剂盒评估的 OA 对细胞活力影响的结果如图 2 所示。计算出 OA 处理的 HepG2 细胞的 GI50</sub...

作者聚焦:建立 MASLD 细胞模型以研究 Koumiss 的疾病机制和降脂作用

本文介绍了使用油酸诱导的 HepG2 细胞作为代谢功能障碍相关脂肪性肝病的模型。

代谢功能障碍相关脂肪性肝病中脂肪沉积的体外建模

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

in-vitro-modeling-fat-deposition-metabolic-dysfunction-associated

Research

JoVE Journal

Biology

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

731 次观看.  Inner Mongolia Medical University. 我们的研究方向是通过建立代谢功能障碍相关的脂肪性肝病细胞模型来研究传统蒙古医学。研究了 koumiss 疗法的作用和机制。存在研究的结果将在未来被证明是有利的，能够建立代谢功能障碍相关的脂肪性肝病细胞模型和研究疾病的潜在机制。未来将基于代谢功能障碍相关脂肪性肝病细胞模型进行研究，以探讨 koumiss 降脂作用的作用机制。

视频: 作者聚焦:建立 MASLD 细胞模型以研究 Koumiss 的疾病机制和降脂作用

使用 3D 荧光脂滴重建评估肝脂肪变性

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

作者聚焦： 使用 3D 重建分析荧光染色脂滴用于肝脂肪变性评估  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

科研

生物化学

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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&amp;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.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

医学

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks 

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

生物学

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

免疫与感染

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

分化肝细胞中的血管性脂细胞病的诱导和特征

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

体外实验性硬化模型:脂质超载介质中的肝细胞培养

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

非酒精性脂肪性肝炎小鼠模型中肝细胞外基质的 3D 成像

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

代谢功能障碍相关脂肪性肝病中脂肪沉积的体外建模

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此视频中的章节

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

优化分析

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

分化肝细胞中的血管性脂细胞病的诱导和特征

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

体外实验性硬化模型:脂质超载介质中的肝细胞培养

非酒精性脂肪性肝炎小鼠模型中肝细胞外基质的 3D 成像