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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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。

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		<tr>
			<td>0.22 &#181;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 &#181;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 cm2 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 &#38; Instrument Co.</td>
			<td>02270113</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8 assays</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>CA1210-1000T</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>NUAIRE</td>
			<td>NU-5710E</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;DMSO Dimethyl sulfoxide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</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 &#38; Technology Co.,Ltd.&#160;</td>
			<td>G1262</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid&#160;</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）。

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a range of conditions, including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma1,2,3,4,5,6, all attributed to factors other than alcohol consuption7. MASLD is the most prevalent liver disease caused by metabolic liver injury, affecting nearly one-quarter of the global population8,9,10,11,12. While the precise pathogenesis of MASLD has not yet been elucidated, various theories attempt to explain its development. One prevailing notion suggests a departure from the classic &#34;two-hit&#34; theory towards a &#34;multiple-hit&#34; model1. Central to these hypotheses is the role of insulin resistance, which is believed to be pivotal in MASLD pathogenesis13. Research indicates that insulin resistance in hepatocytes leads to increased levels of free fatty acids, subsequently forming triglycerides stored within the liver14,15.
Researchers have used both in vivo and in vitro models to simulate fat deposition in MASLD; yet fully replicating its pathomechanism remains challenging. Despite this limitation, these models have been instrumental in studying potential therapeutic targets for MASLD. However, the development of a stable model of MASLD is crucial. While animal models are effective, they are time-consuming and expensive, thus highlighting the growing interest in in vitro cellular models. These models often use single or multiple free fatty acids such as oleic acid (OA) and palmitic acid to recreate diet-induced MASLD. Among these, the human hepatoblastoma cell line HepG2 is often used to establish in vitro cellular models of MASLD.
OA induction stimulates HepG2 cells to replicate fatty deposition akin to MASLD, a method with a well-established history. The aim of this study was to demonstrate the viability, Oil Red O (ORO) staining, lipid content, and triglyceride (TG) level of HepG2 cells treated with 0.25 mM OA. The objective of this experiment was to provide further evidence for the development of MAFLD modeling studies.

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

代谢功能障碍相关脂肪性肝病中脂肪沉积的体外建模

Our research direction is the study of traditional Mongolian medicine by establishing a metabolic dysfunction-associated steatotic liver disease cell model. The role and the mechanism of koumiss therapy are studied. The findings of the presence study will prove advantageous in the future, enabling the establishment of metabolic dysfunction-associated steatotic liver disease cell models and the investigation of the disease underlying mechanisms.A future study will be conducted based on the metabolic-dysfunction associated steatotic liver disease cell model to investigate the mechanism of action of the lipid-lowering effect of koumiss.

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...

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。

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

213 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。