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（插入哺乳动物表达载体）和2μl脂质体2000的脂质体转染的协议。我们选择使用设计用于在哺乳动物细胞中表达了pcDNA 3.3骨干。 </li><li>或者，经过6小时的潜伏期，交流转媒体描述在脂质体转染协议正常HEK293媒体的。 </li><li>为了评价表达的转染PROTE，转染组织培养井的一个子集可用于免疫荧光检测所表达的蛋白质或免疫印迹分析蛋白的表达，用标准技术。 </li></ol> 2。可选固定HEK293细胞转染蛋白表达转染HEK293细胞，可以轻轻地固定，以促进共培养介质中可能是有害的生活HEK293细胞中（ 如下面的步骤3.9），或者允许一次全部板前几个实验的高效制备（见讨论）。 <ol><li>进行试验研究，以确定在HEK293细胞中的蛋白质的表达的时间过程。 </li><li>在转染的HEK293细胞层的，吸媒体从细胞转染后的时间点与最高的表达水平相关。如果发生这种情况的第一个24小时内，不转染后吸出，直到24小时。 </li><li>洗净HE然后轻轻地用D-PBS K293细胞加入500微升4％多聚甲醛（PFA），并在室温下孵育30分钟。 （4％PFA的解决方案，16％的溶液在1X PBS开始用1X PBS稀释1:4）。 </li><li>使用无菌PBS，轻轻洗细胞3次，并培育在PBS O / N在4°C。 </li><li>洗涤细胞3次，用无菌PBS，再加入PBS和离开，在4°C，直到需要时（最大存储时间可能取决于对蛋白质的表达。存储HEK293细胞的转染表达的neuroligin亚型长达2周没有显着变化，观察效果胰岛素分泌）。 </li></ol> 3。 INS-1β细胞共培养细胞系的<ol><li>准备INS-1，加入到500毫升的RPMI 1640（葡萄糖和酚红，无谷氨酰胺）：媒体50 5毫升100X毫升胎牛血清，青霉素/链霉素，5毫升100X L-谷氨酰胺溶液，5毫升丙酮酸钠，500微升两性霉素B，500微升β-巯基乙醇（这是几乎相同的Medi这个通常用于除加入β-巯基乙醇）原代胰岛培养。 </li><li>使用手机汽提塔（或其他的非酶细胞去除），收获INS-1细胞在约70-80％汇合的折扣的T75培养瓶的底部。 48口井，每个烧瓶中提供大致足够的细胞。 </li><li>纺丝后，细胞在离心3分钟，在1200 rpm，重悬在50/50的组合，INS-1和HEK媒体。对于70-80％汇合烧瓶INS-1细胞，重悬在约25毫升混合介质。 </li><li>如果使用HEK293细胞中已修复的多聚甲醛，而不是现场HEK293细胞，INS-1细胞悬浮在100％INS-1媒体而非混合介质。 </li><li>用10毫升移液管，从撞出细胞沉淀，使均匀的细胞悬浮液。 </li><li>吸移除媒体HEK293细胞。 </li><li>使用P1000吸管，轻轻INS-1细胞悬液加500微升到HEK细胞层两旁Øf为好。每6口井后，用10毫升吸管再次悬浮INS-1细胞，以确保细胞悬液均匀。共培养整体方案中的设置在图1中示出。 </li><li>细胞孵育24-48小时，取决于实验的协议。 </li><li>如果超过48小时共培养需要花多长时间，使用可选的步骤，在上述第2 HEK293细胞来修复。 </li><li>如果使用主解离的啮齿动物或人类胰岛[ 例如，参见事先协议朱庇特11-15]，确保一个适当的介质，如RPMI用5ml笔/链球菌，5毫升L-过剩，0.5毫升两性霉素-B是用来代替INS-1媒体。虽然可以使用分离的胰岛细胞在24孔板，在实践中，因为潜在需要大量的胰岛，建议应考虑缩减到48孔板（每口井需要较少的胰岛细胞）。在48孔板，加入约100个​​小岛值得的解离泰德细胞每盘启动。根据对药效的离解，所使用的方法，可能需要较少的胰岛。 </li></ol> 4。胰岛素分泌<ol><li>垫上预温育细胞在250μl的2.5 mM葡萄糖的Krebs-林格碳酸氢盐缓冲液（KRB）最少1小时。要垫上预温育，删除共存培养基，并立即更换KRB。这种交流应该做了一个很好的时间，以尽量减低环境氧INS-1细胞。吸用一只手吹打低（2.5毫米）与其他葡萄糖KRB。 </li><li>外汇预孵育的KRB到250μl的2.5毫米或20 mM葡萄糖的KRB缓冲液以及在一个时间。 </li><li>如果适合于特定的实验中，可能会增加额外泌剂，如100μMIBMX，以产生一个更强大的刺激的胰岛素分泌反应。特别是游离主胰岛β细胞反应不佳的葡萄糖浓度增加ALONE，所以在这里特别IBMX或其他泌此外应考虑16,17。 </li><li>经过1小时的潜伏期，媒体转移到离心管和旋转1500 XG 5分钟。 </li><li>取出200μl的上清液，并使用该胰岛素RIA或ELISA法进行分析。 </li><li>以制备细胞裂解液，将100μlRIPA细胞裂解缓冲液（150 mM氯化钠，1.0％IGEPALCA-630或诺乃P-40，0.5％脱氧胆酸钠，0.1％SDS的50mM Tris，pH 8.0）中与蛋白酶抑制剂的加板取出后，立即媒体和孵化，在4℃20分钟。 </li><li>自旋向下的溶胞产物在10,000×g 15分钟，并取出50微升上清液胰岛素RIA或ELISA分析。 </li></ol>

<ol>
	<li>Kelly, C., McClenaghan, N. H., Flatt, P. 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. Metab. 11, 159-167 (2009).</li><li>Poitout, V., Olson, L. K., Robertson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-secreting+cell+lines:+classification,+characteristics+and+potential+applications.">Insulin-secreting cell lines: classification, characteristics and potential applications.</a> Diabetes Metab. 22, 7-14 (1996).</li><li>Craig, A. M., Graf, E. R., Linhoff, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+central+synapse:+clues+from+cell+culture.">How to build a central synapse: clues from cell culture.</a> Trends in Neurosciences. 29, 8-20 (2006).</li><li>Scheiffele, P., Fan, J., Choih, J., Fetter, R., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin+expressed+in+nonneuronal+cells+triggers+presynaptic+development+in+contacting+axons.">Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons.</a> Cell. 101, 657-669 (2000).</li><li>Abderrahmani, A., 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=Neuronal+traits+are+required+for+glucose-induced+insulin+secretion.">Neuronal traits are required for glucose-induced insulin secretion.</a> FEBS Lett. 565, 133-138 (2004).</li><li>Lowe, A. 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>

我们什么都没有透露。

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

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

coculture-analysis-extracellular-protein-interactions-affecting

Research

JoVE Journal

Medicine

12.8K 次观看.  University of California, San Diego. 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。

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

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

提高在社区医院静脉注射胰岛素政府

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

人掌腱膜挛缩的<em&gt;离体</em&gt;文化为肌成纤维细胞的研究和细胞外基质相互作用

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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

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 is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

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 ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

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

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

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

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

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

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

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

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

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

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

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 ...