Name: coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells
Uploaded: 2013-06-15T14:00:00
Description: Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

这项工作得到国家卫生部授予的R01DK080971和青少年糖尿病研究基金会的资助37-2009-44研究院。我们也感谢来自加州大学圣地亚哥分校儿科糖尿病研究中心（PDRC）的支持。

1。转HEK293层的<ol><li>准备HEK293细胞培养基，加入至500ml的瓶的DMEM（4.5克/毫升的葡萄糖和酚红，无谷氨酰胺）：50毫升胎牛血清，5毫升100X青霉素/链霉素溶液，5毫升100倍的L-谷氨酰胺的溶液和500μl的两性霉素B. </li><li>板出在24孔板使用0.5毫升每口井的HEK293媒体的HEK293细胞。确保细胞均匀地分布在盘底。 </li><li>当达到100％汇合的转染HEK293细胞，用0.8μg的质粒的编码所述感兴趣的蛋白质（插入哺乳动?...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+islet+structure+and+cellular+interactions+in+the+control+of+insulin+secretion.">Role of islet structure and cellular interactions in the control of insulin secretion.</a> Islets. 3, 41-47 (2011).</li><li>Konstantinova, I., 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=EphA-Ephrin-A-mediated+beta+cell+communication+regulates+insulin+secretion+from+pancreatic+islets.">EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets.</a> Cell. 129, 359-370 (2007).</li><li>Jain, R., Lammert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interactions+in+the+endocrine+pancreas.">Cell-cell interactions in the endocrine pancreas.</a> Diabetes Obes. 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W., Madeddu, L., Kelly, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+secretory+granules+and+neuronal+synaptic+vesicles+have+three+integral+membrane+proteins+in+common.">Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common.</a> The Journal of Cell Biology. 106, 51-59 (1988).</li><li>Suckow, A. 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=Expression+of+neurexin,+neuroligin,+and+their+cytoplasmic+binding+partners+in+the+pancreatic+beta-cells+and+the+involvement+of+neuroligin+in+insulin+secretion.">Expression of neurexin, neuroligin, and their cytoplasmic binding partners in the pancreatic beta-cells and the involvement of neuroligin in insulin secretion.</a> Endocrinology. 149, 6006-6017 (2008).</li><li>Suckow, A. 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=Transcellular+neuroligin-2+interactions+enhance+insulin+secretion+and+are+integral+to+pancreatic+beta+cell+function.">Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic beta cell function.</a> The Journal of Biological Chemistry. 287, 19816-19826 (2012).</li><li>Kohnert, K. D., Hehmke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+suspensions+of+pancreatic+islet+cells:+a+comparison+of+methods.">Preparation of suspensions of pancreatic islet cells: a comparison of methods.</a> J. Biochem. Biophys. Methods. 12, 81-88 (1986).</li><li>Qi, 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=Human+pancreatic+islet+isolation:+Part+I:+digestion+and+collection+of+pancreatic+tissue.">Human pancreatic islet isolation: Part I: digestion and collection of pancreatic tissue.</a> J. Vis. Exp. (27), e1125 (2009).</li><li>Qi, 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=Human+pancreatic+islet+isolation:+Part+II:+purification+and+culture+of+human+islets.">Human pancreatic islet isolation: Part II: purification and culture of human islets.</a> J. Vis. Exp. (27), e1343 (2009).</li><li>Szot, G. L., Koudria, P., Bluestone, 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=Murine+pancreatic+islet+isolation.">Murine pancreatic islet isolation.</a> J. Vis. Exp. (7), e255 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J. Vis. Exp. (50), e2096 (2011).</li><li>Hauge-Evans, A. C., Squires, P. E., Persaud, S. J., Jones, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cell-to-beta-cell+interactions+are+required+for+integrated+responses+to+nutrient+stimuli:+enhanced+Ca2++and+insulin+secretory+responses+of+MIN6+pseudoislets.">Pancreatic beta-cell-to-beta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2+ and insulin secretory responses of MIN6 pseudoislets.</a> Diabetes. 48, 1402-1408 (1999).</li><li>Spiess, Y., Smith, M. A., Vale, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superfusion+of+dissociated+pancreatic+islet+cells+attached+to+Cytodex+beads.">Superfusion of dissociated pancreatic islet cells attached to Cytodex beads.</a> Diabetes. 31, 189-193 (1982).</li><li>Pham, P. L., 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=Transient+gene+expression+in+HEK293+cells:+peptone+addition+posttransfection+improves+recombinant+protein+synthesis.">Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.</a> Biotechnol. Bioeng. 90, 332-344 (2005).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+a+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: a vehicle for the expression of recombinant proteins.</a> J. Pharmacol. Toxicol. Methods. 51, 187-200 (2005).</li><li>Maurisse, R., 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=Comparative+transfection+of+DNA+into+primary+and+transformed+mammalian+cells+from+different+lineages.">Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.</a> BMC Biotechnol. 10, 9 (2010).</li></ol>

在共存这里描述的方法提供了一种有效的方法，以确定特定的β-细胞表面的跨膜蛋白的生理重要性，特别是其胞外结构域。通过培养beta细胞或胰岛素瘤细胞（如这里的INS-1细胞）的HEK293细胞的接触显示感兴趣的在细胞表面上的蛋白质，可设计实验，以确定影响细胞外的蛋白质 - 蛋白质相互作用，而无需直接扰乱的细胞的细胞内环境。由于感兴趣的蛋白质表达的HEK293细胞，可以介导的转染的细胞内?...

我们在这里描述的方法，以方便调查特定的跨膜蛋白的胞外结构域如何影响胰岛素的分泌。的方法探测感兴趣的蛋白质与蛋白质（或可能是其他分子）的胰腺β-细胞表面上的相互作用的影响。该方法允许调查的β细胞或其他邻近的细胞（ 如内皮细胞，神经细胞，胰腺α细胞）表达的细胞表面蛋白如何影响β细胞功能的通过跨细胞相互作用（ 即通过与相互作用伙伴的相互作用的表面上相邻的β细胞）。 的细胞的质膜中包含一系列复杂的结构和功能的蛋白质作为细胞外环境的桥梁。通过形成跨细胞连接或塑料信号事件开始，细胞表面蛋白之间的相互作用，可以帮助协调周边小区的功能。胰岛β细胞胰岛内聚集在一起，并以协调的方式采取行动，以维持葡萄糖稳 ​​态1。所揭示的，例如，通过外EPHA ephrinA的neuroligin-2相互作用的葡萄糖刺激的胰岛素分泌的调节的重要性，它正成为越来越清晰的是，增加细胞外的蛋白质之间的相互作用的表面上相邻的知识β细胞，将是非常重要的胰岛素分泌，胰岛β细胞功能的成熟和维持血糖稳态1-3获得一个全面的了解。这里描述的方法的目标是使跨细胞的相互作用，涉及特定的跨膜区或其它细胞表面相关蛋白的β细胞功能的影响的调查。 β细胞共培养HEK293细胞转染不同的表达结构，对b的影响〔η细胞的功能不同的细胞表面蛋白或突变的变种，其可以有效地探测。这是完成，无需转染β细胞本身。 阐明的角色击倒，击倒或过度表达β细胞培养或体内的研究，特别是跨细胞相互作用，必须直接微扰β细胞的mRNA和蛋白表达，有可能影响β细胞的健康和/或功能的方式，可能会混淆分析特定的细胞外的相互作用的影响。这些方法也改变的细胞内结构域的目标蛋白的水平，还不允许或跨细胞的相互作用的影响，以区别于在同一单元中的蛋白质之间的相互作用的影响。在这里，一种方法，用于确定特定的跨细胞的相互作用的效果的β细胞分泌胰岛素的能力和响应能力是叙述的离子束辅助沉积。本方法适用分泌胰岛素的β-细胞系，如INS-1细胞4，并解离的主要啮齿动物或人类β细胞。它是基于共培养模型的神经生物学家，谁发现，暴露培养的神经细胞的特定的神经元蛋白表达对HEK293细胞层（或COS）开发可以识别该驱动器突触形成的蛋白质5,6。由于神经突触和β细胞分泌机械之间的相似之处，我们的理由是，β细胞功能和功能成熟，都可能被类似的跨细胞相互作用7-9。为了探测这些相互作用，我们开发了本文描述的系统，其中beta细胞层上的感兴趣的蛋白10表达的HEK293细胞共培养。这个系统允许的β-细胞的细胞质中保持不变，而细胞外的蛋白质 - 蛋白质相互作用被操纵。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>pcDNA 3.3 vector/backbone</td>
			<td>Invitrogen</td>
			<td>K830001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>18324012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech</td>
			<td>45001-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/strep solution</td>
			<td>Mediatech</td>
			<td>45001-652-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Mediatech</td>
			<td>45001-808-1/30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Mediatech</td>
			<td>45001-404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>45001-434</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Mediatech</td>
			<td>45001-710-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell stripper</td>
			<td>Mediatech</td>
			<td>45000-668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>BD</td>
			<td>1368065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Mediatech</td>
			<td>45001-130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Mediatech</td>
			<td>MT35010CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879-100MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells

细胞表面蛋白之间的相互作用有助于协调相邻细胞的功能。胰岛β细胞胰岛细胞内聚集在一起，并以协调的方式采取行动，以维持血糖稳态。它正变得越来越清楚，相邻的β细胞表面的跨膜蛋白之间的相互作用是β细胞功能的重要决定因素。 阐明的角色击倒，击倒或过度表达β细胞培养或体内的研究，特别是跨细胞相互作用必须直接扰动mRNA和蛋白表达，可能会影响β-细胞的健康和/或功能的方式，可能混淆的影响分析具体的相互作用。这些方法还可以改变对象的蛋白质的细胞内结构域的水平，并可能防止蛋白质之间的相互作用的影响，由于在同一小区内的膜显示tinguished跨细胞的相互作用的影响。 这里，提出了一种用于确定特定的跨细胞的相互作用的效果的β细胞分泌胰岛素的能力和响应能力。此方法适用于β-细胞系，如INS-1细胞，并解离的主要beta细胞。共培养模型的基础上开发的神经生物学家，谁培养的神经元，以表达对HEK293细胞层（或COS）确定蛋白质的重要​​驱动突触形成的特定的神经元蛋白的发现，暴露。由于神经突触和β细胞分泌机械之间的相似之处，我们的理由是，类似的跨细胞相互作用，都可能被β-细胞功能成熟。我们开发了一种系统beta细胞上培养HEK293细胞中表达的蛋白质还一层。在这个模型中，β-细胞的细胞质，而细胞外的蛋白质 - 蛋白质interactio不变NS被操纵。虽然我们对葡萄糖刺激的胰岛素分泌的研究主要集中在这里，其他进程可以进行分析，例如，由免疫或定量PCR基因表达的变化。

使用这里介绍的方法，我们已经测试的效果不同的变种，对胰岛素分泌的蛋白质的neuroligin。这补充了我们的neuroligin-2 10β细胞功能的影响调查工作。例如， 图2中 ，示出从INS-1 beta细胞共培养的结果与转染的HEK293细胞来表达的neuroligin亚型这里称为NL十这个实验的目的是检验这一假设，NL-X从事跨细胞的相互作用，增加胰岛素的分泌。在图2A中可以看出，基底和NL-X的?...

Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

跨细胞蛋白质的相互作用是胰岛β细胞功能的重要决定因素。这里是详细的方法改编自突触调查特定的跨膜蛋白如何影响胰岛素的分泌模式的共存。转染的HEK293细胞中表达蛋白质的利益;不需要beta细胞被转染的或以其他方式直接扰动。

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

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Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

神经变性慢性淀粉样蛋白-β低聚物输注的动物模型是由抗体治疗灌注抵消与渗透泵

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

端到端吻合技术在机器人中心胰腺中的应用

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Homogeneous Time-resolved FÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...