Name: an adipocyte cell culture model to study the impact of protein and micro-rna modulation on adipocyte function
Uploaded: 2021-05-04T14:00:00
Description: Watch this Scientific Journal Video about An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function at JoVE.com

这项工作得到了国家航空研究局、蔚蓝海岸大学和法国国家研究局（ANR）的支持，该计划旨在为未来的卓越实验室（Labex SIGNALIFE-ANR-11-LABX-0028-01）和卓越倡议（Idex UCAJEDI ANR-15-IDEX-0001）进行投资。J.J.得到法语国家协会、法语国家协会、法国多组织技术协会（非国大）和本杰明-德莱塞特基金会的赠款支持。J.G. 得到 ANR-18-CE14-0035-01 的支持。J-F.T. 得到ANR赠款ADIPOPIEZO-19-CE14-0029-01和梅迪卡莱基金会（埃基佩·弗鲁姆，DEQ20180839587）的赠款的支持。我们还感谢由阿尔卑斯-海洋部和Région PACA资助的C3M成像核心设施，该设施也得到了GIS IBISA显微镜和成像平台Céte d'Azur（MICA）的支持。

注：使用无菌技术在层压流动细胞培养罩中执行协议的所有步骤。有关所有试剂和设备的详细信息，请参阅 材料表 。
1. 将穆林 3T3-L1 成纤维细胞分化为脂肪细胞
<ol><li>在培养中DMEM的100毫米菜肴中种植3T3-L1成纤维细胞，不含丙酸酯、25mM葡萄糖、10%新生儿小腿血清和1%青霉素和链霉素（图1A）。将盘子放在组织培养孵化器（7% CO2 和 37...

<ol>
	<li>Kl&#246;ting, N., 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=Insulin-sensitive+obesity.">Insulin-sensitive obesity.</a> American Journal of Physiology, Endocrinology and Metabolism. 299 (3), 506-515 (2010).</li><li>Weyer, C., Foley, J. E., Bogardus, C., Tataranni, P. A., Pratley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+subcutaneous+abdominal+adipocyte+size,+but+not+obesity+itself,+predicts+type+II+diabetes+independent+of+insulin+resistance.">Enlarged subcutaneous abdominal adipocyte size, but not obesity itself, predicts type II diabetes independent of insulin resistance.</a> Diabetologia. 43 (12), 1498-1506 (2000).</li><li>Bl&#252;her, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+dysfunction+contributes+to+obesity+related+metabolic+diseases.">Adipose tissue dysfunction contributes to obesity related metabolic diseases.</a> Best Practice &amp; Research Clinical Endocrinology &amp; Metabolism. 27 (2), 163-177 (2013).</li><li>Lorente-Cebri&#225;n, S., Gonz&#225;lez-Muniesa, P., Milagro, F. I., Mart&#237;nez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+other+non-coding+RNAs+in+adipose+tissue+and+obesity:+emerging+roles+as+biomarkers+and+therapeutic+targets.">MicroRNAs and other non-coding RNAs in adipose tissue and obesity: emerging roles as biomarkers and therapeutic targets.</a> Clinical Science. 133 (1), 23-40 (2019).</li><li>Jager, J., 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=Tpl2+kinase+is+upregulated+in+adipose+tissue+in+obesity+and+may+mediate+interleukin-1beta+and+tumor+necrosis+factor-{alpha}+effects+on+extracellular+signal-regulated+kinase+activation+and+lipolysis.">Tpl2 kinase is upregulated in adipose tissue in obesity and may mediate interleukin-1beta and tumor necrosis factor-{alpha} effects on extracellular signal-regulated kinase activation and lipolysis.</a> Diabetes. 59 (1), 61-70 (2010).</li><li>Vergoni, B., 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=DNA+damage+and+the+activation+of+the+p53+pathway+mediate+alterations+in+metabolic+and+secretory+functions+of+adipocytes.">DNA damage and the activation of the p53 pathway mediate alterations in metabolic and secretory functions of adipocytes.</a> Diabetes. 65 (10), 3062-3074 (2016).</li><li>Berthou, F., 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+Tpl2+kinase+regulates+the+COX-2/prostaglandin+E2+axis+in+adipocytes+in+inflammatory+conditions.">The Tpl2 kinase regulates the COX-2/prostaglandin E2 axis in adipocytes in inflammatory conditions.</a> Molecular Endocrinology. 29 (7), 1025-1036 (2015).</li><li>Ceppo, F., 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=Implication+of+the+Tpl2+kinase+in+inflammatory+changes+and+insulin+resistance+induced+by+the+interaction+between+adipocytes+and+macrophages.">Implication of the Tpl2 kinase in inflammatory changes and insulin resistance induced by the interaction between adipocytes and macrophages.</a> Endocrinology. 155 (3), 951-964 (2014).</li><li>Jager, J., Gr&#233;meaux, T., Cormont, M., Le Marchand-Brustel, Y., Tanti, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta-induced+insulin+resistance+in+adipocytes+through+down-regulation+of+insulin+receptor+substrate-1+expression.">Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression.</a> Endocrinology. 148 (1), 241-251 (2007).</li><li>Jager, J., 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=Tpl2+kinase+is+upregulated+in+adipose+tissue+in+obesity+and+may+mediate+interleukin-1beta+and+tumor+necrosis+factor-{alpha}+effects+on+extracellular+signal-regulated+kinase+activation+and+lipolysis.">Tpl2 kinase is upregulated in adipose tissue in obesity and may mediate interleukin-1beta and tumor necrosis factor-{alpha} effects on extracellular signal-regulated kinase activation and lipolysis.</a> Diabetes. 59 (1), 61-70 (2010).</li><li>Hart, 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=miR-34a+as+hub+of+T+cell+regulation+networks.">miR-34a as hub of T cell regulation networks.</a> Journal of ImmunoTherapy of Cancer. 7, 187 (2019).</li><li>Brandenburger, T., 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=MiR-34a+is+differentially+expressed+in+dorsal+root+ganglia+in+a+rat+model+of+chronic+neuropathic+pain.">MiR-34a is differentially expressed in dorsal root ganglia in a rat model of chronic neuropathic pain.</a> Neuroscience Letters. 708, 134365 (2019).</li></ol>

本文对成熟脂肪细胞的分化和转染提出了详细的协议。这种反向转染方法是一种简单、经济、高效的方法，可将寡核苷酸（如但不限于siRNA、微RNA模仿和抗微RNA）转化为3T3-L1脂肪细胞，这是最难转染的细胞系之一。此方法有一些需要考虑的限制。此协议对于质粒DNA的转染效率不高，这限制了该技术对功能收益研究的效用。虽然包括3T3-L1细胞系在内的紫外细胞系通常用于研究体外脂肪细胞功能，但?...

肥胖被认为是许多代谢性疾病的主要危险因素，包括胰岛素抵抗（IR）、2型糖尿病（T2D）和心血管疾病1。目前的治疗方法未能阻止这些疾病的发病率持续上升，肥胖和糖尿病患者的IR管理仍然是一个重要的临床问题。脂肪组织在控制能量平衡方面起着至关重要的作用，其在肥胖期间的病理扩张有助于红外和T2D2、3的发展。这突出表明需要更好地了解脂肪细胞功能障碍所涉及的分子机制，以开发针对肥胖相关疾病的新疗法。许多研究已经调查了蛋白质编码RNA在脂肪细胞生理学中的作用及其与肥胖的关系。
最近，非编码RNA（ncRNA）的发现，特别是微RNA（miRs），已经锻造了与基因表达程序调控机制相关的新概念。研究表明，ncRNA是脂肪细胞功能的重要调节器，其调节不良在代谢疾病中起着重要作用。因此，蛋白和ncRNA在脂肪细胞中的操纵对于破译它们在脂肪细胞功能中的作用及其对T2D等病理学的影响至关重要。然而，操纵蛋白质和ncRNA在体内和原发性脂肪细胞中的表达仍然极具挑战性，有利于使用体外脂肪细胞模型。
Murine 3T3-L1 成纤维细胞很容易分化成成熟、功能和胰岛素响应的脂肪细胞，这是用于研究脂肪细胞功能（如胰岛素信号、葡萄糖吸收、脂解和脂肪素分泌）5、6、7、8、9、10的具有良好特征的细胞系。这些特性使3T3-L1脂肪细胞成为调节蛋白质编码和nc-RNA表达的有吸引力的模型，以破译它们在脂肪细胞功能中的作用及其在肥胖相关疾病中的潜在作用。不幸的是，虽然3T3-L1成纤维细胞很容易使用商用试剂进行转染，但差异化的3T3-L1脂肪细胞是最难转染的细胞系之一。这就是为什么许多操纵3T3-L1细胞基因表达的研究都集中在脂肪细胞分化上，而不是脂肪细胞功能上。
长期以来，唯一有效的转染脂肪细胞的技术是电聚变5，这是乏味的，昂贵的，并可能导致细胞损伤。本文使用一种常见的转染试剂报告了反向转染技术，它减少了实际转染时间，对细胞生存能力没有影响，而且比电透电便宜得多。此协议非常适合硅酸和其他寡核苷酸（如微RNA模拟（miR模仿）和反微核素的转化。反向转化协议的原则是孵化转染试剂和寡核苷酸，在细胞培养板中形成复合物，然后将成熟的脂肪细胞播种到井中。然后，在寡核苷酸/转染试剂复合物的存在下，脂肪细胞重新连接到粘附板表面。这种简单、高效和廉价的方法允许研究蛋白质编码RNA和miR在脂肪细胞功能中的作用及其在肥胖相关疾病中的潜在作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 well Tissue Culture Plate</td>
			<td>Dutscher</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsin (10x)</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>diluted to 5x with D-PBS</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Accell Non-targeting Pool</td>
			<td>Horizon Discovery</td>
			<td>D-001910-10-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I from calf skin</td>
			<td>Sigma-Aldrich</td>
			<td>C8919</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s&#160; Modified Eagles&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>41965062</td>
			<td>4.5 g/L D-Glucose; L-Glutamine; no Pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>51976</td>
			<td></td>
		</tr>
		<tr>
			<td>FAM-labeled Negative Control si-RNA</td>
			<td>Invitrogen</td>
			<td>AM4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HSP90 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-131119</td>
			<td>Dilution : 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Improved Minimal Essential Medium (Opti-MEM)</td>
			<td>Gibco</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Human Recombinant</td>
			<td>Gibco</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr>
			<td>miRIDIAN micro-RNA mimics</td>
			<td>Horizon Discovery</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript II RT Kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript Primer Assays Hs_RNU6-2_11</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript Primer Assays Mm_miR-34a_1</td>
			<td>Qiagen</td>
			<td>MS00001428</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR Kit</td>
			<td>Qiagen</td>
			<td>219073</td>
			<td></td>
		</tr>
		<tr>
			<td>Newborn Calf Serum</td>
			<td>Gibco</td>
			<td>16010-159</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>ON-TARGETplus Mouse Plin1 si-RNA SMARTpool</td>
			<td>Horizon Discovery</td>
			<td>L-056623-01-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin and Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perilipin-1 antibody</td>
			<td>Cell Signaling</td>
			<td>3470</td>
			<td>Dilution : 1/1000</td>
		</tr>
		<tr>
			<td>Petri dish 100 mm x 20 mm</td>
			<td>Dutscher</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>PKB antibody</td>
			<td>Cell Signaling</td>
			<td>9272</td>
			<td>Dilution : 1/1000</td>
		</tr>
		<tr>
			<td>PKB Phospho Thr308 antibody</td>
			<td>Cell Signaling</td>
			<td>9275</td>
			<td>Dilution : 1/1000</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent (INTERFERin)</td>
			<td>Polyplus</td>
			<td>409-10</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin antibody</td>
			<td>Sigma aldrich</td>
			<td>T6199</td>
			<td>Dilution : 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Vamp2 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB5136</td>
			<td>Dilution : 0.1 &#181;g/mL</td>
		</tr>
	</tbody>
</table>

an adipocyte cell culture model to study the impact of protein and micro-rna modulation on adipocyte function

脂肪细胞功能的改变有助于代谢疾病的发病机制，包括2型糖尿病和胰岛素抵抗。这突出表明需要更好地了解脂肪细胞功能障碍所涉及的分子机制，以开发针对肥胖相关疾病的新疗法。调节脂肪细胞 中 蛋白质和微RNA的表达仍然极具挑战性。本文描述了一种将肉质成成熟脂肪细胞的方案，并利用小干扰RNA（siRNA）和微型RNA模拟（miR模仿）寡核苷酸，通过反向转化调节成熟脂肪细胞中蛋白质和微RNA的表达。此反向转化协议涉及转染试剂和寡核苷酸的孵化，以形成细胞培养板中的复合物，并添加成熟腺细胞。然后，在存在寡核苷酸/转染试剂复合物的情况下，允许脂肪细胞重新连接到粘附板表面。可对转染的3T3-L1成熟脂肪细胞进行胰岛素信号、葡萄糖吸收、脂生成和脂质分析等功能分析，以研究蛋白质或微RNA操作对脂肪细胞功能的影响。

使用此处描述的反向转化程序来调节 3T3-L1 脂肪细胞中蛋白质或微RNA 的表达，已证明这些脂肪细胞在转染后保留了其形态（图 1B，C）。事实上，在转染后2天，脂肪细胞传播良好，并附着在板上，并呈现了成熟3T3-L1脂肪细胞的特性多叶脂液滴。血脂含量在转染和未转染的脂肪细胞之间没有区别（图1D，E）。</st...

Watch this Scientific Journal Video about An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function at JoVE.com

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

这里介绍的是一个协议，将小干扰RNA（siRNA）、微RNA模拟（miR）或抗微RNA（抗miR）等寡核苷酸输送到成熟的脂肪细胞中，以调节蛋白质和微RNA表达。

研究蛋白质和微RNA调制对脂肪细胞功能的影响的脂肪细胞培养模型

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

此协议使在差异化脂肪细胞中操纵蛋白质或微RNA表达来研究它们在脂肪细胞功能中的作用成为可能。这种反向转化方法是将寡核苷酸转化为小鼠3T3-L1脂肪细胞的简单、经济、高效的方法。这种方法也可以应用于区分成脂肪细胞的人类前细胞。演示这个程序的将是梅兰妮·高德夫林，一个来自我们实验室的技术员。在 DMEM 的 100 毫米菜肴中种植 3T3-L1 成纤维细胞，不含皮鲁瓦酸、25 毫摩尔葡萄糖、10% 新生儿小腿血清和 1% 青霉素和链霉素。将盘子放在37摄氏度和7%的二氧化碳的组织培养孵化器中。汇合两天后 改变培养基，代之以不含甲基苯丙胺的DMEM、25毫摩尔葡萄糖、10%FCS、1%青霉素和链霉素，辅以0.25毫摩尔IBMX、0.25微摩尔脱氧三聚氰胺、每毫升胰岛素5微克和10微摩尔松糖酮。将菜肴孵化两天。两天后，用不含皮鲁瓦酸的DMEM、25毫摩尔葡萄糖、10%FCS和1%的青霉素和链霉素代替培养基，每毫升胰岛素补充5微克，再孵育10微摩尔葡萄糖酮，再孵育两天。每两天用DMEM喂养细胞，不含丙酸酯、25毫摩尔葡萄糖、10%FCS和1%青霉素和链霉素，并将细胞保留在孵化器中。在转染前一天或几个小时，从库存溶液中以每毫升 1 毫克 30% 乙醇 100 微克的速度准备胶原蛋白 I 型溶液。每井加入250微升胶原蛋白，每井加入124井板，将溶液铺在井面上。将盘子放在培养罩下没有盖子，直到胶原蛋白干燥。然后用 D- pbs 洗两次。皮佩特siRNA与改进的最低基本介质混合和孵育在室温下五分钟。将转染试剂和改进的极小必需介质添加到 siRNA 和移液器中混合。在室温下孵育20分钟。然后将转染混合物添加到胶原蛋白涂层板的每个井中。用 D-PBS 清洗 100 毫米培养皿中的细胞两次。在细胞中加入5倍试用素，确保用试金素覆盖整个表面。等待 30 秒，并小心地取出试金素。在37摄氏度的孵化器中孵育培养皿5至10分钟。然后点击盘子分离细胞，避免细胞损伤。加入10毫升不含皮鲁瓦酸的DMEM、25毫摩尔葡萄糖、10%FCS和1%青霉素和链霉素，以中和胰蛋白酶。小心地上下移液，分离细胞并使细胞悬架均质化。使用马拉塞兹计数室或自动细胞计数器对细胞进行计数，并将细胞浓度调整为每毫升中等0.625万细胞。每井12井板的细胞悬浮800微升种子，24井板含有转染混合物的每井细胞悬浮400微升。在细胞培养孵化器中孵育板，24小时不打扰它们。第二天，小心地用不含皮鲁瓦酸、25毫摩尔葡萄糖、10%FCS和1%青霉素和链霉素的超高纳坦代替超高纳坦，并将细胞返回孵化器。在 siRNA 或 miR 交付后检测目标击倒的典型时间点为 mRNA 24 到 48 小时，蛋白质的检测时间点为 48 到 96 小时。功能实验可以在转染的脂肪细胞上进行，包括胰岛素信号、葡萄糖吸收、脂肪素分泌、脂解和脂肪生成。染菌术细胞在转染后已证明可以保持其形态。转染两天后，脂肪细胞呈现多细胞脂质液滴，其脂质含量在转染和未转染的脂肪细胞之间没有区别。与未受感染的腺细胞相比，各种分化标记的mRNA表达在转染细胞中保持不变。反向转染协议是有效的，因为超过 70% 的脂肪细胞被转染。在与西里平-1的siRNA转染三天后，佩里平-1的mRNA水平下降了70%，蛋白质水平下降了63%，佩里平-1表达在转染后四天也通过荧光显微镜进行了分析，发现与控制腺细胞的表达相比下降了92%。此处显示了用微RNA模拟寡核苷酸来调节 miR-34a 表达的脂肪细胞的反向转染。miR-34a的过度表达导致VAMP2蛋白表达减少50%，3T3-L1脂肪细胞中佩里平-1的击倒导致基底脂质溶解增加，而miR-34a的过度表达导致胰岛素诱导蛋白激酶B磷酸化和葡萄糖吸收的抑制。重要的是要达到高水平的脂肪细胞分化，对新分化的脂肪细胞进行转染，并仔细监测脂肪细胞与试丁核素的处理。转染后，可以对脂肪细胞进行功能实验，以研究蛋白质或微RNA操作对胰岛素信号、葡萄糖吸收、脂肪生成和脂解的影响。

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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

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

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 ...