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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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）。

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

571 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

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.

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 ...

科研

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

分离人原代瓣膜细胞进行体外疾病建模

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

体外 使用小鼠胚胎干细胞进行血管疾病建模和药物测试的血管生成的三维发芽测定

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

用于体外评估药物诱导肝毒性的人肝脏微生理系统

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...